Thyroid fine needle aspiration molecular assay

Abstract

 The present invention relates to methods, compositions and articles directed to diagnosing thyroid carcinoma, differentiating between thyroid carcinoma and benign thyroid diseases, testing indeterminate thyroid fine needle aspirate samples of thyroid nodules, and determining patient protocols and outcomes.

Description

BACKGROUND OF THE INVENTION

[0001] There are approximately 25,600 new cases of thyroid carcinoma diagnosed in the United States each year, and 1,400 patients will die of the disease. About 75% of all thyroid cancers belong to the papillary thyroid carcinoma type. The rest consist of 10% follicular carcinoma, 5% to 9% medullary thyroid cancer, 1% to 2% anaplastic cancer, 1% to 3% lymphoma, and less than 1% sarcoma and other rare tumors. Usually a lump (nodule) in the thyroid is the first sign of thyroid cancer. There are 10 to 18 million people in US with a single thyroid nodule, and approximately 490,000 become clinically apparent each year. Fortunately only about 5% of these nodules are cancerous.

[0002] The commonly used method for thyroid cancer diagnosis is fine needle aspiration (FNA) biopsy. FNA samples are examined cytologically to determine whether the nodules are benign or cancerous. The sensitivity and specificity of FNA range from 68% to 98%, and 72% to 100% respectively, depending on institutions and doctors. Unfortunately, in 25% of the cases the specimens are either inadequate for diagnosis or indeterminable by cytology. In current medical practice, patients with indeterminate results are sent to surgery, with consequence that only 25% have cancer and 75% end up with unnecessary surgery. A molecular assay with high sensitivity and a better specificity (higher than 25%) would greatly improve current diagnostic accuracy of thyroid cancer, and omit unnecessary surgery for non-cancerous patients.

[0003] Comparative genomic hybridization (CGH), serial analysis of gene expression (SAGE), and DNA microarray have been used to identify genetic events occurring in thyroid cancers such as loss of heterozygosity, up and down gene regulation, and genetic rearrangements. PAX8 and PPARγ genetic rearrangement event has been demonstrated to be associated with follicular thyroid cancer (FTC). Rearrangement of the ret proto-oncogene is related to papillary thyroid cancer (PTC). Down-regulation of thyroid peroxidase (TPO) gene is observed in both FTC and PTC. Galectin-3 was reported to be a candidate marker to differentiate malignant thyroid neoplasms from benign lesions. However, there are other studies demonstrating that Galectin-3 is not a cancer-specific marker. Many genes purported to be useful in thyroid cancer diagnosis lack the sensitivity and specificity required for an accurate molecular assay.

SUMMARY OF THE INVENTION

[0004] The present invention encompasses methods of diagnosing thyroid cancer by obtaining a biological sample from a patient; and measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer.

[0005] The present invention encompasses methods of differentiating between thyroid carcinoma and benign thyroid diseases by obtaining a sample from a patient; and measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid carcinoma.

[0006] The present invention encompasses methods of testing indeterminate thyroid fine needle aspirate (FNA) thyroid nodule samples by: obtaining a sample from a patient; and measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer.

[0007] The present invention encompasses methods of determining thyroid cancer patient treatment protocol by: obtaining a biological sample from a thyroid cancer patient; and measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are sufficiently indicative of cancer to enable a physician to determine the type of surgery and/or therapy recommend to treat the disease.

[0008] The present invention encompasses methods of treating a thyroid cancer patient by obtaining a biological sample from a thyroid cancer patient; and measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of cancer; and treating the patient with a thyroidectomy if they are cancer positive.

[0009] The present invention encompasses methods of cross validating a gene expression profile for thyroid carcinoma patients by: a. obtaining gene expression data from a statistically significant number of patient biological samples; b. randomizing sample order; c. setting aside data from about 10% - 50% of samples; d. computing, for the remaining samples, for factor of interest on all variables and selecting variables that meet a p-value cutoff (p); e. selecting variables that fit a prediction model using a forward search and evaluating the training error until it hits a predetermined error rate; f. testing the prediction model on the left-out 10-50% of samples; g. repeating steps c., -g. with a new set of samples removed; and h. continuing steps c) -g) until 100% of samples have been tested and record classification performance.

[0010] The present invention encompasses methods of independently validating a gene expression profile and gene profiles obtained thereby for thyroid carcinoma patients by obtaining gene expression data from a statistically significant number of patient biological samples; normalizing the source variabilities in the gene expression data; computing for factor of interest on all variables that were selected previously; and testing the prediction model on the sample and record classification performance.

[0011] The present invention encompasses a method of generating a posterior probability score to enable diagnosis of thyroid carcinoma patients by: obtaining gene expression data from a statistically significant number of patient biological samples; applying linear discrimination analysis to the data to obtain selected genes; and applying weighted expression levels to the selected genes with discriminate function factor to obtain a prediction model that can be applied as a posterior probability score.

[0012] The present invention encompasses methods of generating a thyroid carcinoma prognostic patient report and reports obtained thereby, by obtaining a biological sample from the patient; measuring gene expression of the sample; applying a posterior probability thereto; and using the results obtained thereby to generate the report.

[0013] The present invention encompasses compositions containing at least one probe set selected from the group consisting of: SEQ ID NOs: 36, 53, 73, 211 and 242; and/or SEQ ID NOs: 199, 207, 255 and 354; or the psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

[0014] The present invention encompasses kits for conducting an assay to determine thyroid carcinoma diagnosis in a biological sample containing: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

[0015] The present invention encompasses articles for assessing thyroid carcinoma status containing: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

[0016] The present invention encompasses microarrays or gene chips for performing the methods provided herein.

[0017] The present invention encompasses diagnostic/prognostic portfolios containing isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25 where the combination is sufficient to characterize thyroid carcinoma status or risk of relapse in a biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 

Figure 1 is an ROC curve of the LOOCV of the 4-gene signature in 98 training samples.

Figure 2 is an ROC curve of the 5-gene signature in 98 training samples.

Figure 3a is an ROC curve of the 4-gene signature in 74 independent validation samples; 3b is an ROC curve of the 5-gene signature in 74 independent validation samples.

Figure 4a is an ROC curve of the 4-gene signature that is normalized to the three-thyroid control genes; 4b is an ROC curve of the 5-gene signature that is normalized to the three-thyroid control genes.

Figure 5a is an ROC curve of the 4-gene signature with one-round amplification in 47 thyroid samples; 5b is an ROC curve of the 4-gene signature with two-round amplification in 47 thyroid samples; 5c is an ROC curve of the 5-gene signature with one-round amplification in 47 thyroid samples; 5d is an ROC curve of the 5-gene signature with two-round amplification in 47 thyroid samples.

Figures 6a and 6b depict the ROC curves for cross validation with the 83 independent fresh frozen thyroid samples.

Figures 7a and 7b depict the ROC curves for signature validation with the 47 fine needle aspirate (FNA) thyroid samples.

Figures 8a and 8b depict the ROC curves for signature performance in 28 paired fresh frozen and FNA thyroid samples.

DETAILED DESCRIPTION

[0019] In this study the goal was to identify signatures that can be used in assays such as DNA chip-based assay to differentiate thyroid carcinomas from benign thyroid diseases. 31 primary papillary thyroid tumors, 21 follicular thyroid cancers, 33 follicular adenoma samples, and 13 benign thyroid diseases were analyzed by using the Affymetrix human U133A Gene Chip. Comparison of gene expression profiles between thyroid cancers and benign tissues has enabled us to identify two signatures: a 5-gene signature identified by percentile analysis and manual selection, and a 4-gene signature selected by Linear Discrimination Analysis (LDA) approach. These two signatures have the performance of sensitivity/specificity 92%/70% and 92%/61%, respectively, and have been validated in 74 independent thyroid samples. The results presented herein demonstrate that these candidate signatures facilitate the diagnosis of thyroid cancers with better sensitivity and specificity than currently available diagnostic procedures. These two signatures are suitable for use in testing indeterminate FNA samples.

[0020] By performing gene profiling on 98 representative thyroid benign and tumor samples on Affymetrix U133a chips, we have selected two gene signatures, a 5-gene signature and a 4-gene signature, for thyroid FNA molecular assay. Signatures were selected to achieve the best sensitivity of the assay at a close to 95%. Except for fibronectin and thyroid peroxidase, the other seven genes from the two signatures have not been implicated previously in thyroid tumorogenesis. Both signatures have been validated with an independent 74 thyroid samples, and achieved performance that is equivalent to the one in the 98 training samples. The performances of the two gene signatures are 92% sensitivity and 70%/61% specificity, respectively. When these two signatures are normalized to the specific thyroid control genes the performances are improved relative to the ones of the non-normalized signatures. Furthermore, the signatures performed equivalently with two different target preparations, namely one-round amplification and two-round amplifications. This validation is extremely important for thyroid assays that are FNA samples, which usually contain limited numbers of thyroid cells.

[0021] The mere presence or absence of particular nucleic acid sequences in a tissue sample has only rarely been found to have diagnostic or prognostic value. Information about the expression of various proteins, peptides or mRNA, on the other hand, is increasingly viewed as important. The mere presence of nucleic acid sequences having the potential to express proteins, peptides, or mRNA (such sequences referred to as "genes") within the genome by itself is not determinative of whether a protein, peptide, or mRNA is expressed in a given cell. Whether or not a given gene capable of expressing proteins, peptides, or mRNA does so and to what extent such expression occurs, if at all, is determined by a variety of complex factors. Irrespective of difficulties in understanding and assessing these factors, assaying gene expression can provide useful information about the occurrence of important events such as tumorogenesis, metastasis, apoptosis, and other clinically relevant phenomena. Relative indications of the degree to which genes are active or inactive can be found in gene expression profiles. The gene expression profiles of this invention are used to provide a diagnosis and treat patients for thyroid cancer.

[0022] Sample preparation requires the collection of patient samples. Patient samples used in the inventive method are those that are suspected of containing diseased cells such as cells taken from a nodule in a fine needle aspirate (FNA) of thyroid tissue. Bulk tissue preparation obtained from a biopsy or a surgical specimen and laser capture microdissection are also suitable for use. Laser Capture Microdissection (LCM) technology is one way to select the cells to be studied, minimizing variability caused by cell type heterogeneity. Consequently, moderate or small changes in gene expression between normal or benign and cancerous cells can be readily detected. Samples can also comprise circulating epithelial cells extracted from peripheral blood. These can be obtained according to a number of methods but the most preferred method is the magnetic separation technique described in U.S. Patent 6,136,182. Once the sample containing the cells of interest has been obtained, RNA is extracted and amplified and a gene expression profile is obtained, preferably via microarray, for genes in the appropriate portfolios.

[0023] The present invention encompasses methods of diagnosing thyroid cancer by obtaining a biological sample from a patient; and measuring the expression levels in the sample of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer.

[0024] The present invention encompasses methods of differentiating between thyroid carcinoma and benign thyroid diseases by obtaining a sample from a patient; and measuring the expression levels in the sample of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid carcinoma.

[0025] The present invention encompasses methods of testing indeterminate thyroid fine needle aspirate (FNA) thyroid nodule samples by: obtaining a sample from a patient; and measuring the expression levels in the sample of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer.

[0026] The present invention encompasses methods of determining thyroid cancer patient treatment protocol by: obtaining a biological sample from a thyroid cancer patient; and measuring the expression levels in the sample of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are sufficiently indicative of cancer to enable a physician to determine the type of surgery and/or therapy recommend to treat the disease.

[0027] The present invention encompasses methods of treating a thyroid cancer patient by obtaining a biological sample from a thyroid cancer patient; and measuring the expression levels in the sample of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25; where the gene expression levels above or below pre-determined cut-off levels are indicative of cancer; and treating the patient with thyroidectomy if they are cancer positive.

[0028] The SEQ ID NOs in the above methods can be 36, 53, 73, 211 and 242 or 199, 207, 255 and 354, or 45, 215, 65, 29, 190, 199, 207, 255 and 354.

[0029] The invention also encompasses the above methods containing the steps of further measuring the expression level of at least one gene encoding mRNA: corresponding to SEQ ID NOs: 142, 219 and 309; and/or corresponding to SEQ ID NOs: 9, 12 and 18; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 130, 190 and 276 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 9, 12 and 18 as depicted in Table 25. The invention also encompasses the above methods containing the steps of further measuring the expression level of at least one gene constitutively expressed in the sample.

[0030] Cadherin 3, type 1 (SEQ ID NO: 53) is mentioned in US20030194406; US 20050037439; and US 20040137539. Fibronectin (SEQ ID NO: 242) is mentioned in US6436642 and US20030104419. Secretory granule, neuroendocrine protein 1 (SEQ ID NO: 76) is mentioned in US20030232350; and US20040002067. Testican-1 (SEQ ID NO: 36) is mentioned in US20030108963; and US20050037463. Thyroid peroxidase (SEQ ID NO: 211) is mentioned in US6066449, US20030118553; US20030054571; WO9102061; and WO9856953. Chemokine C (C-C) motif ligand 18 (SEQ ID NO: 354) is mentioned in WO2005005601 and US20020114806. Pulmonary surfactant-associated protein B (SEQ ID NO: 355) is mentioned in US20030219760; and US20030232350. K+ channel beta subunit (SEQ ID NO: 207) is mentioned in US20030096782; and US 20020168638. Putative prostate cancer suppressor (SEQ ID NO: 178) is mentioned in WO2005020784. Bone marrow stromal cell antigen 1 (SEQ ID NO: 142) is mentioned in WO2004040014; and WO2005020784. Leucocyte immunoglobulin-like receptor-6b (SEQ ID NO: 219) is mentioned in US20030060614. Bridging integrator 2 (SEQ ID NO: 309) is mentioned in EP1393776; WO02057414; WO0116158 and US6831063. Cysteine-rich, angiogenic inducer, 61 (SEQ ID NO: 9) is mentioned in W02004030615; and WO9733995. Selenoprotein P, Plasma 1 (SEQ ID NO: 12) is mentioned in US20040241653 and W02005015236. Insulin-like growth factor-binding protein 4 (SEQ ID NO: 18) is mentioned in WO2005015236; WO9203469; WO9203152; and EP0546053.

[0031] In this invention, the most preferred method for analyzing the gene expression pattern of a patient in the methods provided herein is through the use of a linear discrimination analysis program. The present invention encompasses a method of generating a posterior probability score to enable diagnosis of thyroid carcinoma patients by: obtaining gene expression data from a statistically significant number of patient biological samples; applying linear discrimination analysis to the data to obtain selected genes; and applying weighted expression levels to the selected genes with discriminate function factor to obtain a prediction model that can be applied as a posterior probability score. Other analytical tools can also be used to answer the same question such as, logistic regression and neural network approaches.

[0032] For instance, the following can be used for linear discriminant analysis:



where,
I_(psid) = The log base 2 intensity of the probe set enclosed in parenthesis.
d_(CP) = The discriminant function for the cancer positive class
d_(CN) = The discriminant function for the cancer negative class
P_(CP) = The posterior p-value for the cancer positive class
P_(CN) = The posterior p-value for the cancer negative class

[0033] Numerous other well-known methods of pattern recognition are available. The following references provide some examples: Weighted Voting: Golub et al. (1999); Support Vector Machines: Su et al. (2001); and Ramaswamy et al. (2001); K-nearest Neighbors: Ramaswamy (2001); and Correlation Coefficients: van't Veer et al. (2002).

[0034] Preferably, portfolios are established such that the combination of genes in the portfolio exhibit improved sensitivity and specificity relative to individual genes or randomly selected combinations of genes. In the context of the instant invention, the sensitivity of the portfolio can be reflected in the fold differences exhibited by a gene's expression in the diseased state relative to the normal state. Specificity can be reflected in statistical measurements of the correlation of the signaling of gene expression with the condition of interest. For example, standard deviation can be a used as such a measurement. In considering a group of genes for inclusion in a portfolio, a small standard deviation in expression measurements correlates with greater specificity. Other measurements of variation such as correlation coefficients can also be used in this capacity. The invention also encompasses the above methods where the specificity is at least about 40%, at least about 50% and at least about 60%. The invention also encompasses the above methods where the sensitivity is at least at least about 90% and at least about 92%.

[0035] The invention also encompasses the above methods where the comparison of expression patterns is conducted with pattern recognition methods. One method of the invention involves comparing gene expression profiles for various genes (or portfolios) to ascribe diagnoses. The gene expression profiles of each of the genes comprising the portfolio are fixed in a medium such as a computer readable medium. This can take a number of forms. For example, a table can be established into which the range of signals (e.g., intensity measurements) indicative of disease is input. Actual patient data can then be compared to the values in the table to determine whether the patient samples are normal, benign or diseased. In a more sophisticated embodiment, patterns of the expression signals (e.g., fluorescent intensity) are recorded digitally or graphically. The gene expression patterns from the gene portfolios used in conjunction with patient samples are then compared to the expression patterns.

[0036] Pattern comparison software can then be used to determine whether the patient samples have a pattern indicative of the disease. Of course, these comparisons can also be used to determine whether the patient is not likely to experience the disease. The expression profiles of the samples are then compared to the portfolio of a control cell. If the sample expression patterns are consistent with the expression pattern for cancer then (in the absence of countervailing medical considerations) the patient is treated as one would treat a thyroid cancer patient. If the sample expression patterns are consistent with the expression pattern from the normal/control cell then the patient is diagnosed negative for cancer.

[0037] Preferably, levels of up and down regulation are distinguished based on fold changes of the intensity measurements of hybridized microarray probes. A 1.5 fold difference is preferred for making such distinctions (or a p-value less than 0.05). That is, before a gene is said to be differentially expressed in diseased versus normal cells, the diseased cell is found to yield at least about 1.5 times more, or 1.5 times less intensity than the normal cells. The greater the fold difference, the more preferred is use of the gene as a diagnostic or prognostic tool. Genes selected for the gene expression profiles of this invention have expression levels that result in the generation of a signal that is distinguishable from those of the normal or non-modulated genes by an amount that exceeds background using clinical laboratory instrumentation.

[0038] Statistical values can be used to confidently distinguish modulated from non-modulated genes and noise. Statistical tests find the genes most significantly different between diverse groups of samples. The Student's T-test is an example of a robust statistical test that can be used to find significant differences between two groups. The lower the p-value, the more compelling the evidence that the gene is showing a difference between the different groups. Nevertheless, since microarrays measure more than one gene at a time, tens of thousands of statistical tests may be asked at one time. Because of this, one is unlikely to see small p-values just by chance and adjustments for this using a Sidak correction as well as a randomization/permutation experiment can be made. A p-value less than 0.05 by the T-test is evidence that the gene is significantly different. More compelling evidence is a p-value less then 0.05 after the Sidak correction is factored in. For a large number of samples in each group, a p-value less than 0.05 after the randomization/permutation test is the most compelling evidence of a significant difference.

[0039] The present invention encompasses microarrays or gene chips for performing the methods provided herein. The microarrays can contain isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25 where the combination is sufficient to characterize thyroid carcinoma or risk of relapse in a biological sample. The microarray preferably measures or characterizes at least about 1.5-fold over- or under-expression, provides a statistically significant p-value over- or under-expression, or a p-value is less than 0.05. Preferably, the microarray contains a cDNA array or an oligonucleotide array and may contain one or more internal control reagents. One preferred internal control reagent is a method of detecting PAX8 gene expression which can be measured using SEQ ID NOs: 409-411.

[0040] Preferably, an oligonucleotide in the array corresponds to the 3' non-coding region of the gene the expression of which is being measured.

[0041] Another parameter that can be used to select genes that generate a signal that is greater than that of the non-modulated gene or noise is the use of a measurement of absolute signal difference. Preferably, the signal generated by the modulated gene expression is at least 20% different than those of the normal or non-modulated gene (on an absolute basis). It is even more preferred that such genes produce expression patterns that are at least 30% different than those of normal or non-modulated genes.

[0042] Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcriptase PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art and are described in U.S. Patents such as: 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.

[0043] Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying effects such as the onset, arrest, or modulation of uncontrolled cell proliferation. Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same. The product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray. Typically, the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells. A large number of such techniques are available and useful. Preferred methods for determining gene expression can be found in US Patents 6,271,002; 6,218,122; 6,218,114; and 6,004,755.

[0044] Analysis of the expression levels is conducted by comparing such signal intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a diseased tissue can be compared with the expression intensities generated from benign or normal tissue of the same type. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.

[0045] Gene expression profiles can also be displayed in a number of ways. The most common method is to arrange raw fluorescence intensities or ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data are arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color. For example, a ratio less than one (indicating down-regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the spectrum. Commercially available computer software programs are available to display such data including "GENESPRING" from Silicon Genetics, Inc. and "DISCOVERY" and "INFER" software from Partek, Inc.

[0046] Modulated genes used in the methods of the invention are described in the Examples. The genes that are differentially expressed are either up regulated or down regulated in patients with thyroid cancer relative to those with benign thyroid diseases. Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline. In this case, the baseline is the measured gene expression of a benign disease patient. The genes of interest in the diseased cells are then either up regulated or down regulated relative to the baseline level using the same measurement method. Diseased, in this context, refers to an alteration of the state of a body that interrupts or disturbs, or has the potential to disturb, proper performance of bodily functions as occurs with the uncontrolled proliferation of cells. Someone is diagnosed with a disease when some aspect of that person's genotype or phenotype is consistent with the presence of the disease. However, the act of conducting a diagnosis or prognosis includes the determination of disease/status issues such as determining the likelihood of relapse, type of therapy and therapy monitoring. In therapy monitoring, clinical judgments are made regarding the effect of a given course of therapy by comparing the expression of genes over time to determine whether the gene expression profiles have changed or are changing to patterns more consistent with normal tissue.

[0047] Genes can be grouped so that information obtained about the set of genes in the group provides a sound basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice. These sets of genes make up the portfolios of the invention. As with most diagnostic markers, it is often desirable to use the fewest number of markers sufficient to make a correct medical judgment. This prevents a delay in treatment pending further analysis as well unproductive use of time and resources.

[0048] One method of establishing gene expression portfolios is through the use of optimization algorithms such as the mean variance algorithm widely used in establishing stock portfolios. This method is described in detail in US patent publication number 20030194734. Essentially, the method calls for the establishment of a set of inputs (stocks in financial applications, expression as measured by intensity here) that will optimize the return (e.g., signal that is generated) one receives for using it while minimizing the variability of the return. Many commercial software programs are available to conduct such operations. "Wagner Associates Mean-Variance Optimization Application," referred to as "Wagner Software" throughout this specification, is preferred. This software uses functions from the "Wagner Associates Mean-Variance Optimization Library" to determine an efficient frontier and optimal portfolios in the Markowitz sense is preferred. Use of this type of software requires that microarray data be transformed so that it can be treated as an input in the way stock return and risk measurements are used when the software is used for its intended financial analysis purposes.

[0049] The process of selecting a portfolio can also include the application of heuristic rules. Preferably, such rules are formulated based on biology and an understanding of the technology used to produce clinical results. More preferably, they are applied to output from the optimization method. For example, the mean variance method of portfolio selection can be applied to microarray data for a number of genes differentially expressed in subjects with cancer. Output from the method would be an optimized set of genes that could include some genes that are expressed in peripheral blood as well as in diseased tissue. If samples used in the testing method are obtained from peripheral blood and certain genes differentially expressed in instances of cancer could also be differentially expressed in peripheral blood, then a heuristic rule can be applied in which a portfolio is selected from the efficient frontier excluding those that are differentially expressed in peripheral blood. Of course, the rule can be applied prior to the formation of the efficient frontier by, for example, applying the rule during data pre-selection.

[0050] Other heuristic rules can be applied that are not necessarily related to the biology in question. For example, one can apply a rule that only a prescribed percentage of the portfolio can be represented by a particular gene or group of genes. Commercially available software such as the Wagner Software readily accommodates these types of heuristics. This can be useful, for example, when factors other than accuracy and precision (e.g., anticipated licensing fees) have an impact on the desirability of including one or more genes.

[0051] The gene expression profiles of this invention can also be used in conjunction with other non-genetic diagnostic methods useful in cancer diagnosis, prognosis, or treatment monitoring. For example, in some circumstances it is beneficial to combine the diagnostic power of the gene expression based methods described above with data from conventional markers such as serum protein markers (e.g., Cancer Antigen 27.29 ("CA 27.29")). A range of such markers exists including such analytes as CA 27.29. In one such method, blood is periodically taken from a treated patient and then subjected to an enzyme immunoassay for one of the serum markers described above. When the concentration of the marker suggests the return of tumors or failure of therapy, a sample source amenable to gene expression analysis is taken. Where a suspicious mass exists, a fine needle aspirate (FNA) is taken and gene expression profiles of cells taken from the mass are then analyzed as described above. Alternatively, tissue samples may be taken from areas adjacent to the tissue from which a tumor was previously removed. This approach can be particularly useful when other testing produces ambiguous results.

[0052] The present invention encompasses methods of cross validating a gene expression profile and the profiles thus obtained, for thyroid carcinoma patients by: a. obtaining gene expression data from a statistically significant number of patient biological samples; b. randomizing sample order; c. setting aside data from about 10% - 50% of samples; d. computing, for the remaining samples, for factor of interest on all variables and selecting variables that meet a p-value cutoff (p); e. selecting variables that fit a prediction model using a forward search and evaluating the training error until it hits a predetermined error rate; f. testing the prediction model on the left-out 10-50% of samples; g. repeating steps c., -g. with a new set of samples removed; and h. continuing steps c) -g) until 100% of samples have been tested and record classification performance. In this method, preferably, the gene expression data obtained in step h. is represented by genes from those encoding mRNA: corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363; or recognized specifically by the probe sets from psids in Table 25 corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363.

[0053] The present invention encompasses methods of independently validating a gene expression profile and the profiles thus obtained, for thyroid cancer patients by obtaining gene expression data from a statistically significant number of patient biological samples; normalizing the source variabilities in the gene expression data; computing for factor of interest on all variables that were selected previously; and testing the prediction model on the sample and record classification performance. In this method, preferably, the gene expression data obtained in step d. is represented by genes from those encoding mRNA: corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363; or recognized specifically by the probe sets from psids in Table 25 corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363.

[0054] The present invention encompasses methods of generating a posterior probability to enable diagnosis of thyroid carcinoma patients by obtaining gene expression data from a statistically significant number of patient biological samples; applying linear discrimination analysis to the data to obtain selected genes; applying weighted expression levels to the selected genes with discriminate function factor to obtain a prediction model that can be applied as a posterior probability score. For instance, the following can be used for Linear Discriminant Analysis:




where,
I_(psid) = The log base 2 intensity of the probe set enclosed in parenthesis.
d_(CP) = The discriminant function for the cancer positive class
d_(CN) = The discriminant function for the cancer negative class
P_(CP) = The posterior p-value for the cancer positive class
P_(CN) = The posterior p-value for the cancer negative class

[0055] The present invention encompasses methods of generating a thyroid carcinoma diagnostic patient report and reports obtained thereby, by obtaining a biological sample from the patient; measuring gene expression of the sample; applying a posterior probability score thereto; and using the results obtained thereby to generate the report. The report can also contain an assessment of patient outcome and/or probability of risk relative to the patient population.

[0056] The present invention encompasses compositions containing at least one probe set from: SEQ ID NOs: 36, 53, 73, 211 and 242; and/or SEQ ID NOs: 199, 207, 255 and 354; or the psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

[0057] The present invention encompasses kits for conducting an assay to determine thyroid carcinoma diagnosis in a biological sample containing: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25. The SEQ ID NOs. can be 36, 53, 73, 211 and 242, 199, 207, 255 and 354 and 45, 215, 65, 29, 190, 199, 207, 255 and 354.

[0058] Kits made according to the invention include formatted assays for determining the gene expression profiles. These can include all or some of the materials needed to conduct the assays such as reagents and instructions and a medium through which nucleic acid sequences, their complements, or portions thereof are assayed.

[0059] Articles of this invention include representations of the gene expression profiles useful for treating, diagnosing, prognosticating, and otherwise assessing diseases. These profile representations are reduced to a medium that can be automatically read by a machine such as computer readable media (magnetic, optical, and the like). The articles can also include instructions for assessing the gene expression profiles in such media. For example, the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above. The articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. A graphical recordation is one such format. Clustering algorithms such as those incorporated in "DISCOVERY" and "INFER" software from Partek, Inc. mentioned above can best assist in the visualization of such data.

[0060] Different types of articles of manufacture according to the invention are media or formatted assays used to reveal gene expression profiles. These can comprise, for example, microarrays in which sequence complements or probes are affixed to a matrix to which the sequences indicative of the genes of interest combine creating a readable determinant of their presence. Alternatively, articles according to the invention can be fashioned into reagent kits for conducting hybridization, amplification, and signal generation indicative of the level of expression of the genes of interest for detecting cancer.

[0061] The present invention encompasses articles for assessing thyroid carcinoma status containing: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25. The SEQ ID NOs. can be 36, 53, 73, 211 and 242; 199, 207, 255 and 354; or 45, 215, 65, 29, 190, 199, 207, 255 and 354.

[0062] The present invention encompasses diagnostic/prognostic portfolios containing isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes from those encoding mRNA: corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or corresponding to SEQ ID NOs: 199, 207, 255 and 354; or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or recognized specifically by the probe sets from psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25 where the combination is sufficient to characterize thyroid carcinoma status or risk of relapse in a biological sample. Preferably, the portfolio measures or characterizes at least about 1.5-fold over- or under-expression or provides a statistically significant p-value over- or under-expression. Preferably, the p-value is less than 0.05.

[0063] The invention further provides the following numbered embodiments:

1. A method of diagnosing thyroid cancer comprising the steps:

a. obtaining a biological sample from a patient; and

b. measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA:

i. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

ii. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

iii. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

iv. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
wherein the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer.

2. A method of differentiating between thyroid carcinoma and benign thyroid diseases comprising the steps:

a. obtaining a sample from a patient; and

b. measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA:

i. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

ii. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

iii. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

iv. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
wherein the gene expression levels above or below pre-deteimined cut-off levels are indicative of thyroid carcinoma.

3. A method of testing indeterminate thyroid fine needle aspirate (FNA) thyroid nodule samples comprising the steps:

a. obtaining a sample from a patient; and

b. measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA:

i. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

ii. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

iii. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

iv. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
wherein the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer.

4. A method of determining thyroid cancer patient treatment protocol comprising the steps:

a. obtaining a biological sample from a thyroid cancer patient; and

b. measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA:

i. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

ii. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

iii. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

iv. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
wherein the gene expression levels above or below pre-determined cut-off levels are sufficiently indicative of cancer to enable a physician to determine the type of surgery and/or therapy recommended to treat the disease.

5. A method of treating a thyroid cancer patient comprising the steps:

a. obtaining a biological sample from a thyroid cancer patient; and

b. measuring the expression levels in the sample of genes selected from the group consisting of those encoding mRNA:

i. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

ii. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

iii. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

iv. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
wherein the gene expression levels above or below pre-determined cut-off levels are indicative of cancer; and

c. treating the patient with thyroidectomy if they are cancer positive.

6. The method of one of embodiments 1-5 wherein the SEQ ID NOs. are 36, 53, 73, 211 and 242.

7. The method of one of embodiments 1-5 wherein the SEQ ID NOs. are 199, 207, 255 and 354.

8. The method of one of embodiments 1-5 wherein the SEQ ID NOs. are 45, 215, 65, 29, 190, 199, 207, 255 and 354.

9. The method of one of embodiments 1-5 wherein the sample is prepared by a method are selected from the group consisting of fine needle aspiration, bulk tissue preparation and laser capture microdissection.

10. The method of embodiment 9 wherein the bulk tissue preparation is obtained from a biopsy or a surgical specimen.

11. The method of one of embodiments 1-5 further comprising measuring the expression level of at least one gene encoding mRNA:

a. corresponding to SEQ ID NOs: 142, 219 and 309; and/or

b. corresponding to SEQ ID NOs: 9, 12 and 18; or

c. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 130, 190 and 276 as depicted in Table 25; and/or

d. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 9, 12 and 18 as depicted in Table 25.

12. The method of one of embodiments 1-5 further comprising measuring the expression level of at least one gene constitutively expressed in the sample.

13. The method of one of embodiments 1-5 wherein the specificity is at least about 40%.

14. The method of one of embodiments 1-5 wherein the specificity is at least about 50%.

15. The method of one of embodiments 1-5 wherein the specificity is at least about 60%.

16. The method of one of embodiments 1-5 wherein the sensitivity is at least at least about 90%.

17. The method of one of embodiments 1-5 wherein the sensitivity is at least at least about 92%.

18. The method of one of embodiments 1-5 wherein the comparison of expression patterns is conducted with pattern recognition methods.

19. The method of embodiment 18 wherein the pattern recognition methods include the use of a Cox proportional hazards analysis.

20. The method of one of embodiments 1-5 wherein the pre-determined cut-off levels are at least about 1.5-fold over- or under- expression in the sample relative to benign cells or normal tissue.

21. The method of one of embodiments 1-5 wherein the pre-determined cut-off levels have at least a statistically significant p-value over-expression in the sample having thyroid carcinoma cells relative to benign cells or normal tissue.

22. The method of embodiment 21 wherein the p-value is less than about 0.05.

23. The method of one of embodiments 1-5 wherein gene expression is measured on a microarray or gene chip.

24. The method of embodiment 23 wherein the microarray is a cDNA array or an oligonucleotide array.

25. The method of embodiment 24 wherein the microarray or gene chip further comprises one or more internal control reagents.

26. The method of one of embodiments 1-5 wherein gene expression is determined by nucleic acid amplification conducted by polymerase chain reaction (PCR) of RNA extracted from the sample.

27. The method of embodiment 26 wherein said PCR is reverse transcription polymerase chain reaction (RT-PCR).

28. The method of embodiment 27, wherein the RT-PCR further comprises one or more internal control reagents.

29. The method of embodiment 28, wherein the internal control reagent is a method of detecting PAX8 gene expression

30. The method of embodiment 29, wherein PAX8 gene expression is measured using SEQ ID NOs: 409-411.

31. The method of one of embodiments 1-5 wherein gene expression is detected by measuring or detecting a protein encoded by the gene.

32. The method of embodiment 31 wherein the protein is detected by an antibody specific to the protein.

33. The method of one of embodiments 1-5 wherein gene expression is detected by measuring a characteristic of the gene.

34. The method of embodiment 33 wherein the characteristic measured is selected from the group consisting of DNA amplification, methylation, mutation and allelic variation.

35. A method of cross validating a gene expression profile for thyroid carcinoma patients comprising the steps:

a. obtaining gene expression data from a statistically significant number of patient biological samples;

b. randomizing sample order;

c. setting aside data from about 10% - 50% of samples;

d. computing, for the remaining samples, for factor of interest on all variables and selecting variables that meet a p-value cutoff (p);

e. selecting variables that fit a prediction model using a forward search and evaluating the training error until it hits a predetermined error rate;

f. testing the prediction model on the left-out 10-50% of samples;

g. repeating steps c., -g. with a new set of samples removed; and

h. continuing steps c) -g) until 100% of samples have been tested and record classification performance.

36. The method according to embodiment 35 wherein the gene expression data obtained in step h. is represented by genes selected from the group consisting of those encoding mRNA:

a. corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363; or

b. recognized specifically by the probe sets selected from the group consisting of psids in Table 25 corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363.

37. A method of independently validating a gene expression profile for thyroid carcinoma patients comprising the steps:

a. obtaining gene expression data from a statistically significant number of patient biological samples;

b. normalizing the source variabilities in the gene expression data;

c. computing for factor of interest on all variables that were selected previously; and

d. testing the prediction model on the sample and record classification performance.

38. The method according to embodiment 37 wherein the gene expression data obtained in step d. is represented by genes selected from the group consisting of those encoding mRNA:

a. corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363; or

b. recognized specifically by the probe sets selected from the group consisting of psids in Table 25 corresponding to SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363.

39. A gene profile obtained by the method according to embodiment 37 or 38.

40. A method of generating a posterior probability score to enable diagnosis of thyroid carcinoma patients comprising the steps:

a. obtaining gene expression data from a statistically significant number of patient biological samples;

b. applying linear discrimination analysis to the data to obtain selected genes;

c. applying weighted expression levels to the selected genes with discriminate function factor to obtain a prediction model that can be applied as a posterior probability score.

41. The method according to embodiment 40, wherein the linear discriminant analysis is calculated using the equation:




where,
I_(psid) = The log base 2 intensity of the probe set enclosed in parenthesis.
d_(CP) = The discriminant function for the cancer positive class
d_(CN) = The discriminant function for the cancer negative class
P_(CP) = The posterior p-value for the cancer positive class
P_(CN) = The posterior p-value for the cancer negative class

42. A method of generating a thyroid carcinoma prognostic patient report comprising the steps:

a. obtaining a biological sample from the patient;

b. measuring gene expression of the sample;

c. applying a Relapse Hazard Score to the results of step b.; and

d. using the results obtained in step c. to generate the report.

43. The method of embodiment 42 wherein the report contains an assessment of patient outcome and/or probability of risk relative to the patient population.

44. A patient report generated by the method according to embodiment 42.

45. A composition comprising at least one probe set selected from the group consisting of: SEQ ID NOs: 36, 53, 73, 211 and 242; and/or SEQ ID NOs: 199, 207, 255 and 354; or the psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

46. A kit for conducting an assay to determine thyroid carcinoma prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA:

a. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

b. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

c. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

d. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

47. The kit of embodiment 46 wherein the SEQ ID NOs. are 36, 53, 73, 211 and 242.

48. The kit of embodiment 46 wherein the SEQ ID NOs. are 199, 207, 255 and 354.

49. The kit of embodiment 46 wherein the SEQ ID NOs. are 45, 215, 65, 29, 190, 199, 207, 255 and 354.

50. The kit of embodiment 46 further comprising reagents for conducting a microarray analysis.

51. The kit of embodiment 46 further comprising a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

52. Articles for assessing thyroid carcinoma status comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA:

a. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

b. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

c. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

d. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25.

53. The articles of embodiment 52 wherein the SEQ ID NOs. are 36, 53, 73, 211 and 242.

54. The articles of embodiment 52 wherein the SEQ ID NOs. are 199, 207, 255 and 354.

55. The articles of embodiment 52 wherein the SEQ ID NOs. are 45, 215, 65, 29, 190, 199, 207, 255 and 354.

56. The articles of embodiment 52 further comprising reagents for conducting a microarray analysis.

57. The articles of embodiment 52 further comprising a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

58. A microarray or gene chip for performing the method of one of embodiments 1-5.

59. The microarray of embodiment 58 comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA:

a. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

b. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

c. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

d. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
where the combination is sufficient to characterize thyroid carcinoma or risk of relapse in a biological sample.

60. The microarray of embodiment 59 wherein the measurement or characterization is at least about 1.5-fold over- or under-expression.

61. The microarray of embodiment 59 wherein the measurement provides a statistically significant p-value over- or under-expression.

62. The microarray of embodiment 59 wherein the p-value is less than about 0.05.

63. The microarray of embodiment 59 comprising a cDNA array or an oligonucleotide array.

64. The microarray of embodiment 59 further comprising or more internal control reagents.

65. A diagnostic/prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of those encoding mRNA:

a. corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242; and/or

b. corresponding to SEQ ID NOs: 199, 207, 255 and 354; or

c. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 36, 53, 73, 211 and 242 as depicted in Table 25; and/or

d. recognized specifically by the probe sets selected from the group consisting of psids corresponding to SEQ ID NOs: 199, 207, 255 and 354 as depicted in Table 25
where the combination is sufficient to characterize thyroid carcinoma status or risk of relapse in a biological sample.

66. The portfolio of embodiment 65 wherein the measurement or characterization is at least about 1.5-fold over- or under-expression.

67. The portfolio of embodiment 65 wherein the measurement provides a statistically significant p-value over- or under-expression.

68. The portfolio of embodiment 65 wherein the p-value is less than about 0.05.

[0064] The following examples are provided to illustrate but not limit the claimed invention. All references cited herein are hereby incorporated by reference herein.

Example 1

Materials and Methods

Tissue samples

[0065] Fresh frozen thyroid benign diseases, follicular adenoma, follicular carcinoma, and papillary carcinoma samples were obtained from different commercial vendors including Genomics Collaborative, Inc. (Cambridge, MA), Asterand (Detroit, MI), and Proteogenex (Los Angeles, CA). All samples were collected according to an Institutional Review Board approval protocol. Patients demographic and pathology information were also collected. The histopathological features of each sample were reviewed to confirm diagnosis, estimate sample preservation and tumor content.

RNA isolation

[0066] Standard TriZol protocol was used for all the RNA isolations. Tissue was homogenized in TriZol reagent (Invitrogen, Carlsbad, CA). Total RNA was isolated from TriZol and precipitated at -20°C with isopropyl alcohol. RNA pellets were washed with 75% ethanol, dissolved in water and stored at -80°C until use. RNA integrity was examined with Agilent 2100 Bioanalyzer RNA 6000 NanoAssay (Agilent Technologies, Palo Alto, CA).

Linear Discrimination Analysis

[0067] Linear Discriminant Analysis was performed using these steps: calculation of a common (pooled) covariance matrix and within-group means; calculation of the set of linear discriminant functions from the common covariance and the within-group means; and classification using the linear discriminant functions.

[0068] Plugging the chip intensity readings for each probe into the following equation can be used to derive the posterior probability of an unknown thyroid sample as either cancer positive or negative. For example, if a thyroid sample is tested with the assay and gives a p_(CP) > 0.5 this sample will be classified as thyroid cancer.

[0069] For the 4 gene signature:

[0070] For the 5 gene signature:








where,
I_(psid) = The log base 2 intensity of the probe set enclosed in parenthesis.
d_(CP) = The discriminant function for the cancer positive class
d_(CN) = The discriminant function for the cancer negative class
P_(CP) = The posterior p-value for the cancer positive class
P_(CN) = The posterior p-value for the cancer negative class

Two-round aRNA amplification

[0071] aRNA was amplified from 10 ng total RNA using the RiboBeast 2-Round Aminoallyl-aRNA Amplification kit (Epicentre, WI), a T7 based RNA linear amplification protocol, with some modifications. Total RNA was reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence and Superscript III RT. The second-strand synthesis was carried out using Bst DNA polymerase. An extra step of incubation with an exonuclease mix of Exo I and Exo VII was performed to reduce background. The double-stranded cDNA served as the template for T7-mediated linear amplification by in vitro transcription. For the second round of amplification, instead of using the RiboBeast reagents, the ENZO BioArray HighYield RNA Transcript Labeling kit (Affymetrix, CA) was used in place of the in vitro transcription step of Aminoallyl-aRNA. The aRNA was quantified by Agilent Nano Chip technology.

Example 2

Microarray analysis

[0072] Labeled cRNA was prepared and hybridized with the high-density oligonucleotide array Hu133A Gene Chip (Affymetrix, Santa Clara, CA) containing a total of 22,000 probe sets. Hybridization was performed according to a standard protocol provided by the manufacturer. Arrays were scanned using Affymetrix protocols and scanners. For subsequent analysis, each probe set was considered as an independent gene. Expression values for each gene were calculated by using Affymetrix Gene Chip analysis software MAS 5.0. All chips met the following quality control standards: the percentage of "presence" call, the scaling factor, the background level, and the noise level have to be within the range of mean plus or minus 3 standard deviation. All chips used for subsequent analysis have passed these quality control criteria. Sample collection for signature selection and independent validation is summarized in Table 1.

Table 1. Sample collection for signature training and validation

Training Sample Set
Category	Number of Samples
Follicular Adenoma (FA)	33
Follicular Carcinoma (FC)	21
Benign Diseases (BN)	13
Papillary Carcinoma (PC)	31

Validation Sample Set
Category	Number of Samples
Follicular Adenoma (FA)	38
Follicular Carcinoma (FC)	5
Follicular Variant of Papillary Carcinoma (FVPTC)	11
Papillary Carcinoma (PC)	20

Example 3

Results Signature Identification

A. Gene Selection

[0073] A total of 98 samples including 31 primary papillary thyroid tumors, 21 follicular thyroid cancers, 33 follicular adenoma, and 13 benign thyroid tissues were analyzed by using Affymetrix human U133A gene chips. Five gene selection criteria were applied to the entire data set to obtain a limited number of genes for subsequent gene marker or signature identification:

1. Genes with at least one "Present Call" in this sample set were considered.

2. Genes with more than one "Present Call" in 12 PBL samples were excluded.

3. Only genes with chip intensity larger than 200 in all samples were selected.

4. Using genes that passed the above three criteria, we performed a variety of analyses, as listed in Table 2, to identify genes that are either up-regulated or down-regulated in thyroid tumors.

5. Finally, genes with expression change greater than 1.4-fold were selected.

Table 2. Summary of different types of percentile analyses

	Type of Percentile Analysis
1	20% FC vs 100% Benign
2	30% FC vs 90% Benign
3	30% PC vs 90% Benign
4	70% FC vs 50% Benign
5	70% PC vs 50% Benign
6	90% Benign vs 30% FC
7	90% Benign vs 30% PC

[0074] The final number of selected genes for signature identification is 322, described in Table 25, SEQ ID NOs: 1, 4, 7, 8, 10-11, 13-17, 19-24, 26-27, 29-31, 33-35, 37-38, 40-52, 54-72, 75-82, 84-135, 138-141, 144-151, 153-159, 161-162, 164, 166-173, 176-198, 200-201, 203-206, 208-209, 212-213, 215-218, 220-221, 223, 227-233, 235-241, 243-244, 246-249, 251, 253-254, 256-263, 265-289, 291-293, 295-308, 310-331, 333-341, 343-345, 347-348, 350-353 and 355-363. The data obtained from the 322 selected genes are provided in Table 3 and summarized in Table 4.

Table 4. 4-Gene signature performance in 98 training samples

	Tumor	Benign
Positive	48	18
Negative	4	28
Sensitivity	92% (0.82, 0.97)
Specificity	61% (0.46, 0.74)

B. Signature Identification using Linear Discrimination Analysis

[0075] We used a forward selection process that adds one gene at a time until the posterior error as evaluated by a linear discriminator is less than or equal to 0.1. A four-gene signature was discovered using this approach with the 322 genes. The identities of these 4 genes are listed in Tables 5 and 16 and their chip data are shown in Tables 6 and 7.

[0076] 

Table 5. 4-Gene Signature

SEQ ID NO:	Gene Name
354	Chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated)
199	Pulmonary surfactant-associated protein B (SP-B)
207	K+ channel beta subunit
255	Putative prostate cancer tumor suppressor

[0077] Leave One Out Cross Validation (LOOCV) resulted in 92% sensitivity and 61% specificity, shown in Table 4. The ROC curve gave an AUC of 0.897, as shown in Figure 1.

Table 6

	Signal
SEQ ID	PC_984TT	PC_986TT	FA_987TT	PC_988TT	PC_989TT	FA_992TT	FA_993TT
12	3399.2	7041.3	5438	6376.7	2734.6	3569.5	4305.9
18	6550.8	5870.6	5815.9	4265.1	8856.4	3454.4	20405.3
29	4578.6	6455.8	2329.4	4259	2666.8	3694	7531.2

	FA_994TT	FA_995TT	FA_ 996TT	FA_998TT	FA_999TT	FA_1001TT	FA_1002TT
12	2545.7	2451.8	4418	3938.3	3648.5	6834.5	4882
18	12566.4	9889.5	4341.5	5210.1	6639.5	4794.8	3921.9
29	2239.8	1834.5	4607.1	6424.1	3069.6	4301.4	3164

	FA_1004TT	FA_1005TT	FA_1006TT	FA_1010TT	FA_1013TT	FA_1014TT	FA_1017TT
12	2274.9	2644.2	3978.8	2999.7	4012.5	1724	5005.8
18	10402	7376.3	9985.1	1330.3	4618.8	6132	4562.6
29	1688.9	4771.2	3159.1	3082.6	6861.7	1465	3384.7

	FA_1018TT	FA_1020TT	FA_1023TT	FA_1024TT	FA_1026TT	FA_1027TT	FA_1028TT
12	2847.6	7102	7513.5	2086.3	2431.3	1318.2	1231
18	6388.5	2640.8	1967.7	6853.3	5068.5	2006.4	1804.9
29	3829.5	4235	4272.5	924	1702.3	1691.5	1731.3

	FA_1029TT	FA_1030TT	FA_1031TT	FA_1032TT	FA_1034TT	FA_1035TT	FC_1037TT
12	4607.6	2416.1	2203.7	6489.9	2095.3	1547.9	1847.4
18	3507.3	2270.8	8058	11247.5	8258.1	7485.8	8309
29	1530.5	5641.4	3460.1	4802.1	1650.7	854.6	2418.4

	PC_1039TT	PC_1040TT	PC_1041TT	PC_1042TT	PC_1043TT	PC_1044TT	PC_1045TT
12	8204.9	2638.5	4795	2607.8	6725.4	5178.7	2111.1
18	6454.7	10129.3	7964	4952.6	3663.3	1887.7	19840.6
29	8858.5	4136.8	8434	2230.7	3434	2770	2243.1

	PC_1046TT	PC_1047TT	PC_1048TT	PC_1049TT	PC_1050TT	PC_1051TT	PC-FV_1052TT
12	4403.6	9394.7	2262.5	4831.2	2826.9	1288.6	1953.2
18	2417.7	5678.4	6655.2	4325.2	1706	6904.8	4596.1
29	2319.8	20509.6	1176.1	5375.3	2053.1	4027.5	3332.3

	PC 1053TT	PC 1054TT	PC 1055TT	PC 1059TT	PC 1060TT	PC 1061TT	PC_1062TT
12	4150.4	6180.4	1835.7	2447.2	4201.3	4984.3	3399.3
18	5965.7	13847.6	3740.6	4167.7	14516.2	4896.7	8891.6
29	3718.1	4818.3	1728.8	2972.2	4851	3469	3718.6

	PC-FV_1064TT	PC-FV_1065TT	PC-FV_1066TT	PC-FV 1067TT	PC-FV 1068TT	PC-FV_1069TT	PC-FV 1071TT
12	4221	4884.5	7256.6	640	4055	3208.2	6301.3
18	4095	6627.6	1427.1	5405.5	10722	11509.9	10314.3
29	5896.7	5461.2	9437.3	1634.4	1344.2	2186.4	1794.8

	PC-FV_1072TT	FA_1073TT	FA _1074TT	FA-1075TT	FA-1076TT	FC_1077TT	FC_1078TT
12	1390.3	3325.5	1430.8	1784.4	1563.9	1999.4	1830.7
18	5629	10010.5	3459.2	11454	10807.8	1384.8	11906.2
29	2974.5	7133.1	3009	4184.9	1673.9	1044.5	4264.1

	FC_1079TT	PC-FV_1080TT	PC-FV_1081TT	FC_1082TT	pb1	pb2	pb3
12	2856.2	2437.9	3541.2	4439.8	11.4	9.1	9.9
18	22738	4344.1	4108.3	3312.3	94.2	50.7	67.6
29	1092.8	3052.8	3302.6	2383	8.7	21.6	30.5

	pb4	pb5'	pb6'	pb7'	pb8'	pd10	pd11
12	33.8	8.3	6.6	18.8	61.4	38.2	10.7
18	74.9	29.7	53.6	37.8	31.8	68.8	117.5
29	66.1	64.6	36.4	51.5	25.1	17.2	17.2

	pd12	pd9
12	4.1	11.6
18	70.8	23.4
29	19.7	11

Table 7

	Signal
SEQ ID	FA_987TT	FA_992TT	FA_993TT	FA-994TT	FA-995TT	FA_996TT	FA_998TT
354	50.9	100.4	63.7	14	699.7	181.7	11.9
199	81.7	18	43.3	130.3	461.9	469.4	135.6
207	3844.9	1325.1	77.9	397.2	518.3	807.5	1084.3
255	1374.4	2332.1	1631	611.1	1616.7	419.2	230.7

	FA_999TT	FA_1001TT	FA_1002TT	FA_1004TT	FA_1005TT	FA_1006TT	FA_1010TT
354	194.7	2397.3	50.6	205.2	9.6	29.1	341.5
199	124.1	574.9	206	158.5	423.6	121	2489.4
207	1861.5	1325.1	846.6	308.8	1765.9	598.9	1067.7
255	266.5	186.7	324.2	94	1071.3	1098.5	1294

	FA_1013TT	FA_1014TT	FA_1017TT	FA_1018TT	FA_1020TT	FA_1023TT	FA_1024TT
354	207.5	69	21.7	20.1	64.7	87.4	372.5
199	440.8	99.8	221.6	226.9	108.2	155.2	112.6
207	535.7	1910.9	1185.2	571.8	1552.7	2739.5	692.6
255	437.2	655.6	165.1	178.4	86.4	436.5	40.7

	FA_1026TT	FA_1027TT	FA_1028TT	FA_1029TT	FA_1030TT	FA_1031TT	FA_1032TT
354	21.5	18.1	3.8	13.3	66	15.1	38.8
199	70.9	46.1	561.3	67.7	35.8	48	107.3
207	1230.4	80.5	732	3216.4	2253.5	1989.9	2115.2
255	479.9	226.5	35.9	1403.4	1144.3	247.9	1989.4

	FA_1034TT	FA_1035TT	FA_1073TT	FA_1074TT	FA_1075TT	FA_1076TT	PC_984TT
354	61.3	25.3	1355.7	188.5	14.7	8.9	97.5
199	216.5	56.1	1980.6	454.1	86.2	173.8	154.2
207	421.8	1493.5	771.6	1291.4	116.5	1169.9	759.2
255	489.2	229.5	205.3	655	152.7	156.3	152

	PC_986TT	PC_988TT	PC_989TT	PC_1039TT	PC_1040TT	PC_1041 TT	PC_1042TT
354	966.6	619	12.9	2577	540.4	1229.7	320
199	103.9	142.5	288.5	1006.6	3990.7	17402.7	1023.2
207	400.5	2357.5	1213.8	627.5	62.6	133.6	981.5
255	299.7	2137.5	131.9	500.6	2412.4	2763.1	303.4

	PC_1043TT	PC_1044TT	PC_1045TT	PC_1046TT	PC_1047TT	PC_1048TT	PC_1049TT
354	444.3	170.8	817.6	1095.1	966.8	1263.5	800.7
199	604.3	222.7	22961.4	9488.3	10311.6	1702.7	1366
207	1680.3	594.2	136.5	673.2	755	20.8	254.8
255	407.6	4162.4	872.5	2472.3	1497.3	2944.8	2399.9

	PC_1050TT	PC_1051TT	PC_1053TT	PC_1054TT	PC_1055TT	PC_1059TT	PC_1060TT
354	292.9	614.4	1035.4	1545.3	139.5	500.2	37.1
199	117	29233.8	1435	8550.5	242.8	13914.6	2377.3
207	87.8	113.4	73.3	187.7	491.6	158.4	207.3
255	2331.8	917.2	1505.9	2186.4	3033.2	2191.1	215.4

	PC_1061TT	PC_1062TT	FC_1037TT	FC_1077TT	FC_1078TT	FC_1079TT	FC_1082TT
354	71	23.8	18.4	28.9	87.2	13.5	27.7
199	6933.5	616.2	21.2	226.7	200.8	40.8	144.4
207	1088.5	1133.5	838.8	2012.3	24.9	196.8	1356.4
255	3084.8	1017.8	1355.3	199	1352.4	1578	1294.4

	PC-FV_1052TT	PC-FV_1064TT	PC-FV_1065TT	PC-FV_1066TT	PC-FV_1067TT	PC-FV_1068TT	PC-FV_1069TT
354	394.8	131.4	143.7	281.9	302.1	973.6	44.9
199	4620.7	1931	1613.2	170.8	579.2	20066.3	96.9
207	88.2	1033.1	705	244.1	2559	8.8	12.3
255	2479.7	242.2	1322	641.9	1222.8	1914.8	93.2

	PC-FV_1071TT	PC-FV_11072TT	PC-FV_1080TT	PC-FV_1081TT
354	109.5	127.2	205.7	12.4
199	257.5	2302.7	320	206
207	42.9	72.2	2795.9	145
255	1128.1	2320.8	1705.2	196.8

C. Manual Selection of Markers

[0078] Individual genes were selected with an aim to formulate a RT-PCR based assay. Comparison of gene expression profiles between thyroid cancers and non-cancer tissues has identified a five-gene signature from these 322 genes. The identities of these five genes are shown in Tables 8 and 16 and the chip data are shown in Table 9. The performance of this signature was assessed using LDA in the 98 samples, and the signature gives 92% sensitivity and 70% specificity, shown in Table 10. The ROC curve gave an AUC of 0.88, as shown in Figure 2.

Table 8. 5-Gene signature

SEQ ID NO:	Gene Name
53	Cadherin 3, type 1 (P cadherin)
242	Fibronectin
73	Secretory granule, neuroendocrine protein 1
36	Testican-1
211	Thyroid Peroxidase (TPO)

Table 9

SEQ ID	BN_800TT	BN_801TT	BN_802TT	BN_804TT	BN_805TT	BN_806TT	BN_807TT
36	908.3	247	118.9	349.7	425.1	229.8	227.7
53	48.6	25	24.1	34	17.8	32.2	16.7
76	252.3	272.9	75.2	260.6	169	587.1	189.8
242	22247.9	1204.2	676.7	2441.5	3900.1	2073.4	2060.1
211	24.2	16983.5	32579.5	20189.3	24480.5	14186.3	6488.1

	BN_871TT	BN_913TT	BN_914TT	BN_915TT	FA_917TT	FA_918TT	FA_919TT
36	897.2	435.1	442.3	508.7	169.6	184.3	213.3
53	192.4	28	76.1	44.2	67.7	135.7	25.7
76	2134.6	252.5	956.3	174.3	62	144	233.8
242	13722.5	10481.9	4172.9	1891.1	1654.2	11791	2788.1
211	6243.3	16339.1	14355	36350.1	29876.5	19395.7	15935.8

	FA_818TT	FA_819TT	FA_820TT	FA_821TT	FA_822TT	FA_842TT	FA_862TT
36	120.4	155.8	210.6	31.7	283.3	122.2	24.1
53	27.2	26.9	34	33.2	34.4	27.8	66.1
76	13	69.8	36.2	171.2	116.2	190.8	15.9
242	1433.3	2211.8	2714.7	325	2278	3959.5	1573.1
211	15976.6	19882.4	10029.6	29411.3	25179.3	12492.5	32721.4

	FA_863TT	FA_864TT	FA_865TT	FA_866TT	FA_867TT	FA_869TT	FA_907TT
36	955.1	173.1	400.5	230.8	233.5	2581.5	207.8
53	333.9	27.9	147.1	155	25	1224.9	52.8
76	2614.7	624.1	614.9	336.7	163.5	1921.3	38.5
242	930.4	1419.2	4708.4	2169.8	1081.9	3241.5	4555.3
211	19631.3	27382.8	29846	25011.6	30599.1	23680.6	35313.3

	FA_908TT	FA_920TT	FA_938TT	FA_940TT	FA_941TT	Fa_EA4037 4_921TT	Fa_EA40376 _923TT
36	329.6	165.4	331.5	298.8	267.4	211.3	188.6
53	35.8	28.5	21.7	291.4	45.3	23.8	62.8
76	253.8	64.7	3312.7	870.1	1038.3	153.3	236.1
242	1630.4	2452.4	6684	765	2229.5	1809.2	3314
211	21639.5	31897.6	26420.3	18786.4	31922.5	36025.3	15065.2

	BN_EA40377_9 24TT	Fa_EA40378_92 5TT	Fa_EA40379_ 926TT	BN_EA4038 0_927TT	Fa_EA4038 7_955TT	Fa_EA4038 8_956TT	Fa_EA40389 _957TT
36	1409.1	215.1	228.5	833.3	94.7	259.7	786.2
53	2294.2	40.5	496.1	64.9	197.4	230.4	17.3
76	624.1	149.1	211.3	501.1	2225.4	1443.7	223.7
242	21699.6	1079.3	21653.5	3263.3	2194.8	989.6	2107.8
211	664.1	12583.3	13036.6	43191.4	9409.2	19630.9	4160.9

	Fa_EA40390_9 59TT	Fa_EA40391_96 0TT	Fa_EA40392_ 961TT	Fa_EA40393 _962TT	PC_829TT	PC_830TT	PC_831TT
36	2496.1	175.3	58.2	1470.7	330.3	210.3	432.7
53	24.4	40.3	52.6	183	73	49.6	1916.2
76	2397	47.6	77.2	1048.1	758.4	576.2	394.3
242	1991.2	1042.5	1474.6	688.1	6760	6785	25733
211	38927.5	31764	34287.3	44314.7	25565	31930.4	138.4

	PC_832TT	PC_834TT	PC_835TT	PC_836TT	PC_837TT	PC_838TT	PC_839TT
36	1376.8	4076.8	1208.3	585	1015.5	1620.2	71
53	1909.1	2701	1178.5	710.7	1469.6	3009.9	41.2
76	433.6	665.5	654.3	2432.3	412.1	1946.7	327.2
242	9423.8	18037.6	23053.8	1044.8	22442.7	27313.9	3641
211	7174.2	1961.7	282.1	24646.9	15840.2	264.2	17049.3

	PC_879TT	PC_881TT	PC_882TT	PC_883TT	PC_884TT	PC_885TT	PC_886TT
36	457.4	1779.4	561.2	722.1	1129.2	552.5	1244.9
53	2036.8	3121.5	2072.4	2753	2221.1	1703.8	1325.9
76	1168	1829.9	1210.6	2663.6	1076	1232.2	1866.8
242	46283.3	32477.3	33142.3	38026.7	42087.5	49249	39172.5
211	86.8	613.5	48.3	126.2	1565.2	120	1553

	PC_890TT	PC_892TT	PC_893TT	PC_894TT	PC_903TT	PC_904TT	PC_928TT
36	813.3	2211.4	1046.2	585.3	3395	767.9	246.6
53	4685.6	3536.8	1363.5	1244.4	3057.5	199.9	41.1
76	3926.2	2787	2359.3	1020.3	1564	203.6	421.5
242	36114.4	31599.6	34700.3	41193.8	18485.8	19181.5	3729.4
211	1569.2	1703.6	234.5	1594	4063.1	5354.1	18199.2

	PC_932TT	PC_933TT	Pc_EA40375_ 922TT	Pc_EA40381 _945TT	Pc_EA4038 2_946TT	Pc_EA4038 3_947TT	Pc_EA40384 _948TT
36	352.4	920.3	316.8	818.6	3078.8	532.1	311.1
53	3447.4	788.3	41.8	803.9	2330.1	1337.6	1556.2
76	850.2	865.6	211.2	237.9	1830	1279.7	598
242	21913.7	24536	1758.5	13301.5	22076.9	31987.1	27579.2
211	490.5	13171.6	30959.7	331	857.8	2007.7	556.5

	FC_823TT	FC_824TT	FC_825TT	FC_827TT	FC_828TT	FC_840TT	FC_896TT
36	282.5	2413	1506.3	821.8	564.4	331.6	857.1
53	23.2	24.1	25.5	348.1	27.8	32.3	245.4
76	460.1	2833.9	1128.9	1421.6	705.3	341.8	358.9
242	12129	22000.8	874.8	2544.9	845.1	3459.7	1998.1
211	30520.5	496.2	7923.9	11508.2	33401.7	10882.8	1625.3

	FC_898TT	FC_899TT	FC_900TT	FC_901TT	FC_902TT	FC_909TT	FC_910TT
36	827.5	1152.4	778.8	11199.3	578.7	713	132.4
53	38.1	1949.7	2221	29.8	185.9	21.5	57.2
76	2517.5	1009.8	3523.8	28603.2	2201.1	195.6	4744.8
242	39786.8	38117	29127.6	4032.5	43673.6	3478.5	1273.2
211	1160.7	132.9	79.4	26.8	251.4	488	8979.7

	Fc_EA40386_954TT	Fc_EA40394_967TT	Fc_EA40395_968TT	Fc_EA40396_969TT	Fc EA40397_970TT	Fc_EA40405_982TT	Fc_EA40406_983TT
36	619.3	412.7	134	218.6	463	1233.6	234.6
53	15.8	31	23.8	55.9	65.5	31.6	120.8
76	4136.1	682.5	41.2	18.4	594.7	931	2232
242	969.6	5003.3	13867.1	2129.7	15742.7	4086.9	11985.6
211	20581.7	1509.8	2603.1	15427.4	369.3	30161.2	19869.7

Table 10. 5-Gene signature performance in 98 training samples

	Tumor	Benign
Positive	48	14
Negative	4	32
Sensitivity	92% (0.82, 0.97)
Specificity	70% (0.55, 0.81)

D. Cross Validation with the 74 Independent Thyroid Samples

[0079] 74 independent thyroid samples were processed and profiled with the U133a chip, and the chip data for these two signatures are shown in the Table 11. The performances of the 4-gene and the 5-gene signatures were assessed with LDA. Both signatures gave equivalent performance in these samples compared to the 98 training samples. The sensitivity and specificity for both signatures are shown in Table 12, and the ROC curves are demonstrated in Figure 3a and 3b.

Table 11

	Signal
SEQ ID	FA_987TT	FA_992TT	FA_993TT	FA_994TT	FA_995TT	FA_996TT	FA_998TT
36	92.8	437.8	640.5	4152.5	714.7	254.8	303.4
53	15.8	422.8	25.2	29.5	258.9	45.6	26.8
76	485.1	2536.5	1708.6	288.3	643.3	605.6	341
242	686.5	971.4	24532.8	895.7	6424.8	842.1	905.1
211	28269.8	14689.4	357.5	33711.6	34943.2	16131	22376.1

	FA_999TT	FA_1001TT	FA_1002TT	FA_1004TT	FA_1005TT	FA_1006TT	FA_1010TT
36	63.7	22.3	55.6	408.5	128.3	353	442
53	30.9	31.8	39.6	70.8	37.9	38.9	119.6
76	110.7	90.2	253.5	159.1	1559.6	413.7	1222.5
242	1550.6	1689.3	256.5	1668	3921.5	1013.2	1837.3
211	26779.6	15870.7	24525.5	43040.2	25392.9	32641.9	20140.7

	FA_1013TT	FA_1014TT	FA_1017TT	FA_1018TT	FA_1020TT	FA_1023TT	FA_1024TT
36	453.5	333.7	179.6	146.6	417.3	75.1	129.2
53	39.8	21.4	99.9	23.7	26.8	62.3	22.2
76	333.7	1231.2	241.1	131	1435.3	1491.6	260.8
242	1459.4	415	1696.3	287.1	617.5	1932.6	1689.2
211	7911.3	19359.4	18089	18222.8	30038.8	9053	15611.2

	FA_1026TT	FA_1027TT	FA_1028TT	FA_1029TT	FA_1030TT	FA_103HT	FA_1032TT
36	281.1	130.5	150.4	629.1	760.4	191.6	1671.5
53	125.5	62.3	118.2	230.7	420.5	27.7	519
76	77.4	111.9	184	2100.9	1741.3	338.1	3643.5
242	1091.6	754.3	705.9	684.7	372.4	1848.8	3094.7
211	21979.1	7890.5	26226	15344.1	10368	30439.9	23943.3

	FA_1034TT	FA_1035TT	FA_1073TT	FA_1074TT	FA_1075TT	FA_1076TT	PC_984TT
36	24.8	119.9	111.7	1010.9	26	224.5	303
53	29.6	30.4	366.7	96.9	94.3	31	37.2
76	289.3	239.7	167.4	326.8	818.9	248.5	198.3
242	1482.1	590.9	4842.5	2815.5	2726.8	754.8	785.1
211	38135.4	30510.6	29406.4	31075.4	20833.9	34960.4	24938.2

	PC_986TT	PC_988TT	PC_989TT	PC_1039TT	PC_1040TT	PC_1041TT	PC_1042TT
36	676.3	391.7	264.9	735.7	524.7	769.9	1125
53	32.5	395.6	305.5	655	1153.9	2194.1	111.5
76	287.9	1384.8	888.3	1048.4	1381.1	412.6	1384.9
242	2450.7	2243.2	698.6	20366.4	32090.9	27480.9	1915.7
211	2689.5	19903.2	32075.9	9197.7	152.2	1862.2	10375.9

	PC_1043TT	PC_1044TT	PC_1045TT	PC_1046TT	PC_1047TT	PC_1048TT	PC_1049TT
36	1013.6	1023.8	809.8	1811.1	1546.2	481.8	2491.9
53	730.7	2965.3	2533.7	2052.8	756.1	988.3	2000.6
76	1977.4	3380.4	422	4343.9	3570.2	1786.8	1619.4
242	3907.5	20074.8	41761.2	33253.5	28124.7	30516	21324.5
211	7015.5	11.5	136.1	59.1	3841.7	31.1	1476

	PC_1050TT	PC_1051TT	PC_1053TT	PC_1054TT	PC_1055TT	PC_1059TT	PC_1060TT
36	443.9	669	477.1	952.8	645.3	660.2	648.5
53	4037.8	1290.7	933.9	1336.9	2123.2	1353.8	66.9
76	856.7	1039	463.2	1399.8	4385	365.8	160.7
242	1261.6	58258.5	26004.9	21219.8	12760.8	26084.4	3277.6
211	1150.2	375.9	73.2	2575.8	400.5	24.2	1637.7

	PC_1061TT	PC_1062TT	FC_1037TT	FC_1077TT	FC_1078TT	FC_1079TT	FC_1082TT
36	3523.2	1912.5	234.2	128.3	825.1	297.5	1447.6
53	2348.3	975.7	97.6	46.2	15.8	38.5	254.6
76	4994.9	3891.7	5559.8	252	1103.5	700	1754.7
242	15397.4	2006.1	1826	2407.5	22661.8	1595.5	713.7
211	3379.4	2201.9	41570	15804.5	3572.1	4377.9	16863.7

	PC-FV_1052TT	PC-FV_1064TT	PC-FV_1065TT	PC-FV_1066TT	PC-FV_1067TT	PC-FV_1068TT	PC-FV_1069TT
36	1993.3	230.6	1128.8	708.4	1348	331.4	33.2
53	2450.3	72.7	610.3	72.5	146.4	1073.7	21.5
76	2075	451.3	1350.2	922.5	1743.7	950.5	332.8
242	21494	7110.9	10228.8	2206.3	3319.2	28034.2	430.1
211	1581.5	29936.9	1594.7	5693.1	11288.8	573.6	12485.5

	PC- FV_1071TT	PC-FV_1072TT	PC-FV_1080TT	PC-FV_1081TT
36	148.9	332.2	1288.7	672
53	1836.9	1450.3	2221	22.9
76	4053	1628.6	2275.9	62.6
242	1810.6	26396.9	683.4	984
211	8.9	24	3416.7	2630.8

Table 12. 4-Gene and 5-gene signatures performance in 74 validation samples

4-Gene Signature
	Tumor	Benign
Positive	33	12
Negative	3	26
Sensitivity	92% (0.78, 0.97)
Specificity	68% (0.53, 0.81)

5-Gene Signature
	Tumor	Benign
Positive	33	9
Negative	3	29
Sensitivity	92% (0.78, 0.97)
Specificity	76% (0.61, 0.87)

E. Control Gene Marker Identification

[0080] With the 98 thyroid samples and 12 PBL samples we selected two groups of genes as sampling control. One group consists of genes that are expressed in thyroid but not in PBL, the second group includes genes that are expressed in PBL but not in thyroid. The full gene list and corresponding chip data are shown in Tables 13a and 13b. From these genes we selected six genes that are abundant and the differentiation between thyroid and PBL is relatively large. Their expression profile was validated in the 74 independent thyroid samples. The identities of these six genes are listed in Table 14 and their chip data are shown in Tables 15a and 15b.

Table 13a

	13a1
		ThyMixBen_......_Signal
	SEQ ID		02014_40062_H 133A_800TT		02014_40063_H 133A_801TT	02014_40064_ H133A_802TT	02014_40065_ H133A_804TT	02014_40066_ H133A_805TT	02014_40067_ H133A_806TT
	2		2789.6		2676.2	3009.2	2957.5	3644.5
	3		6346.1		794.7	623.9	772	2977.3	622.7
	5		845.5		3039.1	4452.6	2982.5	8283.2	1627.6
	6		2730.4		1108.5	1155.6	2201.2	1954.3	1750
	9		2248.9		12094.5	7529.2	2827.3	7701.7	1548.3
	12		10625.3		3137.2	2382.7	5727.8	6238	4685.1
	18		3171		5947.9	13241.7	10294.9	7849.3	4037.4
	22		2715.2		9522.5	6429.7	2590.9	5294.3	6490.4
	25		19159.3		1467.3	550.1	2838	11294.7	1374.2
	28		722.7		835.1	727.1	568.1	1336.7	740.7
	32		4160.6		8115.2	4061.8	6625.3	7348.9	3964.1
	39		314.3		1243.1	1486.8	742.5	1027.7	1185.9
	74		323.9		1227.6	1965.1	1281.5	760.1	505.4
	78		250.8		677	1029.5	403.7	795.2	355
	143		1337.3		1339.1	2607.9	1758.8	1933.5	1809
	174		1263.1		4430.7	6041.6	3543.2	7346	3920.5
	175		1996.3		1829.5	3632.7	1646.5	2847.5	3054.2
	191		7108.4		848.3	2063	1133.9	1482.4	1149
	212		258.2		1405.8	1196.9	882.4	1296.8	1790
	222		20586.8		1371.2	2411.9	2573.9	1228.6	1342
	233		1410.6		14126.8	3020.4	2186.1	8439.4	6024.4
	234		5048.1		2183.3	3215.5	4103.9	4096.9	2220.2
	237		905		3397.9	2525.1	2135.4	2822	2492.3
	238		198		1937.7	1289.5	804.5	1538.6	1454.7
	245		143.7		1598.5	1964.2	897.9	2276.6	1348.2
	250		1300.9		777	1405.9	1466.4	1301.3	1865.3
	252		945.6		1547.3	1210.2	1357	1437.1	1141.1
	264		455.1		988.1	932.8	631.7	1253.9	845.4
	290		345.6		327.2	533.1	470.2	700.5	231.9
	294		1043		3655.8	4435.4	3563.9	4377.4	2734.8
	296		845.5		1242.5	1068.6	1506.5	2423.4	1527.3
	342		771.4		535.9	998.5	922.5	1108.1	605.4

	13a2
		Signal
		ThyMixBen_0201402014_40198
	SEQ IID		_4006807_TT	H133A_8_T	_H133A_871T	02014_40321_ H133A_913TT	02014_40322_ H133A_914TT	02014_40323_ H133A_915TT	02014_40324_H 133A_917TT
	2		1749.6		1571.1	2634.4	2419.2	2697.4	2901.1
	3		377.2		508	2322.7	259.8	1123.4	875.2
	5		2762.9		1795.8	6670.2	2094.7	11425.7	4957.2
	6		1280.1		2033.3	1910.3	2543.7	2173.2	1358.7
	9		3346 8		548 3	11398 5	953 4	2085 2	10684.4
	12		6927.1		4060.5	6664 4	2730.5	4322.8	2616 2
	18		7418 9		148604	3134 7	26576 3	9585.4	8588 9
	22		1797.5		744 5	7334.3	317 9	5120 1	8611.7
	25		1245.8		815.8	7201.1	253.7	2882.9	1053.4
	28		301.1		640.8	1325.9	409.2	762.7	838.8
	32		2992 4		3260 2	3588 1	792 5	5413.2	2581
	39		435.8		958.1	1152.1	499.6	556.9	898.6
	74		689.2		1453.7	491.7	736.6	986.9	1217
	78		160.1		383	151.2	172.9	597.4	745.6
	143		1338.8		1466.6	1069.9	2070.9	2377.2	1976.3
	174		2464.3		1616.3	829.8	1365.2	4938	4804.3
	175		1701		1511.5	1101.1	2325.4	2530.2	2237.6
	191		763.6		2383.3	2013.2	1274.2	601.2	1100.2
	212		481		1131.9	912.5	663.2	666.4	1081
	222		280.8		1574	4944.1	342.2	1225.9	449
	233		1289.5		1272.4	6285.6	866 8	1839.3	5797.4
	234		1645.4		3271.1	2263.3	1789.3	6497.4	3416
	237		1732.6		879.6	3780	1345.3	2154.2	2802.8
	238		493.5		893.1	1098.7	405.9	592.8	1326.4
	245		661 9		1378 3	1244 1	645 4	11242	1171.1
	250		736.7		1305.6	633	1409.4	1368.9	1146.7
	252		1145.8		1986	1203.2	1323.5	1544.6	757
	264		462.7		891.3	629.4	649.7	872.1	1319.5
	290		295.8		697.1	987.8	819.9	440.6	268.7
	294		1909.1		1128.4	924.1	1519.2	3225.3	2954.9
	296		898.4		1232.2	925.1	1290.3	738.1	1069.6
	342		482.4		712.6	476.1	694.8	801.2	605.7

	13a3
		Signal
						ThyFolBen_0201	ThyFolBen_02014	ThyFolBen_02	ThyFolBen_020
	SEQ ID		02014_40325_ H133A_918TT		02014_40326_ H133A_919TT	4_40079_H133A_ _818TT	40080_H133A_8 19TT	014_40081_H 133A_820TT	14_40082_H13 3A_821TT
	2		3457.7		2769.1	4234.5	5589.2	3035.3	2681.6
	3		920.5		1289.3	627	690.9	585.4	610
	5		1805.2		5184.1	1487.2	1942	2663.8	1231.8
	6		1560.8		1519.3	996.2	1140.3	2093.4	841.6
	9		11354 8		9921.9	3894	2165.4	7467	76856
	12		7531 7		6887 2	1379.1	12924	5085.1	3585 9
	18		8220.1		5452 2	7762.8	7277 4	5374 9	2379 1
	22		4750 8		5739 1	5655 3	7618 6	6227 9	1551 9
	25		1557.9		3017.5	1200.4	881.1	1279.8	406.4
	28		886.6		1189.2	731.4	681.6	1225.7	473.9
	32		4134 1		10964 4	9276	3075 2	2775	608.8
	39		1259.2		1022.1	1423.7	1632.9	899.5	688.9
	74		741.8		948.9	770	674.6	1139.7	213
	78		447.9		488.2	790.6	602.8	776.3	332.1
	143		3165.1		1865.5	2330.7	1160.7	1676	1672.4
	174		607.5		2151.8	4026.2	4702.6	10677.1	3826.8
	175		5358.5		2402.7	3765.6	1231.9	2531.9	2307.7
	191		2685.9		1183.6	1661.4	1657.1	1377.8	368.2
	212		1306.3		1072.1	2181.8	2035.6	954.2	1663.4
	222		2332.8		431.2	1015.6	1651.7	3755.4	1669.9
	233		5181 8		12377	3400 9	4679 3	2476	15066
	234		3760.2		1988.2	3527.4	3971.4	4526.5	997.6
	237		2712		3073.8	1930.6	3521.8	2951.3	2673.5
	238		1552.2		1636.1	1333.5	837.6	1069.7	1860.5
	245		1722.2		1981.5	1978 8	2170	7636	1392 9
	250		1502.7		1094	1083.3	710.8	864.9	1823.2
	252		1609.9		1679.3	1599.4	1463.9	1199.9	1019.2
	264		860.1		835.6	1451.1	1315.6	1221.6	936.8
	290		773.4		490.4	218.3	507.2	648.3	294.6
	294		555.3		2490.7	1854.9	2997.4	7108.2	2160.7
	296		1240.1		1863	1139.6	1245.4	1172.5	1318.3
	342		738.7		509.3	1018.4	728.8	977.2	710.6

	13a4
		Signal
	SEQ ID		ThyFolBen_02014_40 083_H133A_822TT		fa_EA40173_V DX842TT	fa_EA40191_fa_EA40192 VDX862TT	_VDX863TT	fa_EA40193 _VDX864TT	fa_EA40194 _VDX865TT
	2		3403.4		3423.5	2246.6	2723.3	4130.4	2828.3
	3		644.4		950.1	1313.2	344.4	1731.1	2576.9
	5		3439.5		754.3	2119	3324.7	1663.9	4423
	6		1373.7		987.7	834.7	1399.9	1115.4	1872.1
	9		7457 5		2730 9	9307 8	7048 5	3775.7	6931 3
	12		3978 9		9462.8	777.5	4280 8	4821	2268 2
	18		6386.9		1591 2	9546.6	4979 5	5005.2	4409 5
	22		2652 5		7124 7	11054 7	4669 9	4776 9	5782.5
	25		1887.7		470.8	703.9	714.5	445.9	698.2
	28		548.5		562.4	596.8	915.9	1194.1	1041
	32		4425		2120.3	1084 1	2638.9	2593.1	1919.9
	39		1103.1		1110.2	898.4	1375.1	1520.4	1744.1
	74		285.9		895.8	532.1	841.2	1938.6	1109.5
	78		643.1		346.6	533.3	680.5	621.5	385.8
	143		1979.9		3302.9	1686.6	2406.6	2255.4	3419.1
	174		5408.1		608.2	5072.8	5907.1	2956.1	2670.2
	175		2650.3		6596.8	2600.8	4704.8	3123	5286.8
	191		667.7		2665.7	1382.1	740.2	1133.3	3678.2
	212		1357.8		2545.2	1590.2	1326.3	1135.1	1079
	222		2239.9		471	548.8	584.1	899.4	1002.2
	233		7318.7		11617 9	10868.4	6925.9	5416 9	14212.9
	234		2395.9		2832.7	6005.3	2493.8	2128.8	6818.7
	237		2275.3		2639.3	2406.6	2595.1	1857.2	2546.1
	238		1181.8		2124.4	1586.7	2125.3	2039.5	1859.1
	245		2082 5		2302 1	2846 4	2280 8	3973 8	1520 6
	250		1684		1718	862.9	2782.6	1973.4	1594.3
	252		1166.7		1579.6	590.4	1623.2	1735.2	1584.3
	264		654.2		1604.2	3062.9	1460.8	1510.1	2018.9
	290		342		431.8	458.8	602.3	745	977.6
	294		2978.3		651.5	3533.7	4065.9	2720.4	2292.8
	296		2132.4		1762.9	815.3	1040.3	1787.6	932.6
	342		1058.5		488.3	475.4	759.3	658.3	851.2

	13a5
			Signal
	SEQ ID		fa_EA40195_V DX866TT		fa_EA40196_V DT867TT	fa_EA40197_VDX869TT	fa_EA40219 _VDX907TT	fa_EA40220 _VDX908TT	02014_40327_ H133A_920TT
	2		3243.6		2969.9	3761.4	3352.1	2486.7	3109.7
	3		728.9		548.2	531.1	796	1204.6	981.6
	5		2392.7		6268.7	3870.9	10216	3635.2	11319.3
	6		1534		965.4	2782.8	2638.6	1155.7	2100.9
	9		8238.3		8829 9	4175.6	16504	7363.2	3172
	12		33346		2752	3382 5	3589 1	6717 7	3191 1
	18		9146.3		8150.5	7796 3	8021 5	8185	8907 1
	22		4665.5		8163.5	3812	4001.7	5539.5	6346.7
	25		555.6		524.6	1592.8	2057.7	4103.3	886.1
	28		1115.3		604	918.9	1190.9	999.1	998.2
	32		1089.3		1808 7	1245 6	4202	7454.6	1100 2
	39		1606.8		1418.2	1392.4	1443.3	949.8	1388.7
	74		638.2		2085.2	694.7	1778.1	557.7	1486.9
	78		480.6		603.6	959.8	1000.3	476	721.1
	143		3003		2033.2	3441.7	3224.1	2461.3	2701.2
	174		3322		4573.5	4168.2	5982	3022.2	5710.7
	175		4747.7		2573.2	5834.5	4666.3	3540.8	3751
	191		2724.1		726	831.2	1991.3	1556.8	643.4
	212		2362.7		1705.9	2344.8	1259.8	1288.5	1033.6
	222		271.7		2039.4	683.4	1370.8	728.8	1675.2
	233		10892.9		6981 9	10048.5	6773.9	15803	3520.9
	234		4015.4		3229.5	4734.4	7233.3	2712.8	8148.3
	237		2159.9		2415.2	2194.8	2990.7	2565.8	2364
	238		1451		1111.6	1037.4	914.9	1634.9	1078.9
	245		2059 1		25486	2180.9	2093 2	1730	1329 4
	250		3534.3		993.3	2513.3	878.9	686.3	980.4
	252		1589.8		1334.1	2762.4	1267.3	1857.6	1608.2
	264		1335.1		1560.1	1656.9	942.7	950.4	1076.1
	290		496.3		626.2	584.9	549.5	309.7	613.4
	294		2869.4		4842.4	2762.9	4119.2	2299.8	3497.8
	296		1414.4		882.4	1284	1106.4	1493.4	888.5
	342		595.6		660.6	1442.5	1050.3	607.5	1278.6

	13a6
			Signal
	SEQID		02014_40332_ H133A_938TT		02014_40335_ H133A_940TT	02014_40334_H133A_941TT	EA02014_40374 _H133A_921TT	EA02014_40376 _H133A_923TT	EA02014_40377 _H133A_924TT
	2		1963.7		2662.2	2416.5	3047.7	2043.6	3322
	3		942.5		342	749.1	1300.6	549.3	1182
	5		2218		3903.1	3011.1	1669.8	1508.3	1453.3
	6		939.5		1239.9	1853.8	837.7	1477.8	1129.4
	9		5245 3		1988 9	1165 8	4137	8735.7	4453 3
	12		2500.4		2100	3597 7	5716 2	6694.6	3667 8
	18		5929 7		7819 7	53354	4017.5	2899 7	5013 3
	22		2265 8		6298 2	2112 7	3386 6	4519 5	4186 6
	25		1160.2		916.5	614.7	1055.5	1184.4	828.4
	28		617.6		408.5	740.7	477.3	701.3	550.8
	32		2918.8		3242 3	3083 9	1411 4	4079 5	2063 5
	39		1356.6		876.8	1855	1231.3	982.1	1248
	74		1102.1		826.7	1384.4	296.9	632.9	787.7
	78		361.5		383.5	482.7	578.5	351.8	389.9
	143		1394.9		2889.2	1838.1	1776	1563	3881.9
	174		2198.3		1887.5	3921.8	3076.4	1858.1	1967.3
	175		1391.4		3677.8	2169.3	2169	2397.2	5896.8
	191		1235.1		364.7	1283.5	616.4	1037.7	1957
	212		1625.4		871.1	1083.8	1086.2	1450.7	981.1
	222		503.4		638.5	320.1	1707.8	1089.4	640.8
	233		8499.4		1999.9	4012 1	3345.8	10991 6	11095 5
	234		2067.7		2973.7	2630.4	2027.1	1597	3240
	237		2763.1		1501.1	2724	1997.9	2249.3	2941.1
	238		1055.6		1055.7	1596.5	1035.6	1810.9	1257.5
	245		751.4		1076 1	9152	27144	2120 1	1553.6
	250		1050.6		1474.3	2517.1	1428.1	1169.4	2857.1
	252		987.8		1382.5	2070.7	881.9	1589.3	2217.2
	264		1278.3		1261.1	826.2	865.3	1055.9	1034.7
	290		786.4		936.8	952.1	312.4	385.7	1231.1
	294		2197.5		2064.7	2836.2	2573.5	1835.4	2657.6
	296		635.2		572.8	700.9	1768.2	1426.3	2462.5
	342		718.6		866.8	735.7	770.7	565.3	1695.1

	13a7
		EA02014_ Signal
	SEQ ID		40378_H133A _925TT		40379_H133A_ 926TT	40380_H133A_9 27TT	40387_H133A_9 55TT	40388_H133A_9 56TT	40389_H133A_9 57TT
	2		3274		3025.4	2424.9	2302.6	2724.6	2641.3
	3		1545.1		1151.1	687.6	424.7	420.6	609.2
	5		3661.8		1323.2	7949.9	4860	4059.3	4895.3
	6		1371.5		1220.8	1831	2330.9	1642.8	1963.8
	9		10387.7		11670 1	6402 7	3105 3	4256 6	8744 8
	12		5829.9		6903 8	4783 9	2874 5	3494 1	2451
	18		6648		4053 1	7410.2	4600 2	3912 9	16235 3
	22		6859.7		6621 2	3008 1	1369.9	2480 5	4685
	25		1035.6		3448.2	1347.1	365.3	1587.2	816.8
	28		1087		917.1	747.8	1057	1169.8	974.1
	32		5212 9		6841 6	3282.5	28142	4083 1	8121 2
	39		1228.7		1245.7	762.4	904.9	1136.2	1862.7
	74		823.9		721.6	1460.4	2781.4	970.2	839
	78		713.8		377.6	621.3	212.8	421.3	891.4
	143		1953.5		2925.7	2861.8	1243.3	2335.1	2564.3
	174		3029		1818.4	5264.1	3925.2	4789.1	4553.5
	175		3229.1		4827.2	3821.1	1561.7	4603.8	4098.3
	191		1112.4		5308.2	841	861.6	865.2	1964.9
	212		890.9		915.6	729.1	1264.1	1494.2	1177.2
	222		480.1		2895.3	605.6	145.2	425.3	1712.4
	233		6680 7		9190 5	2236 9	441 5	3626.6	11326 8
	234		1871.2		4194.1	4428.8	1260.8	1599	4627.7
	237		2758.4		2108.7	2070.1	1655.9	2140.3	2820.6
	238		1210.7		1289.5	774.3	1017.9	1645.6	1150.6
	245		2369 7		1585.8	785 1	1808 4	2014 9	1185 6
	250		971.8		1387.3	1193.5	2014.8	2033	608.6
	252		1417.1		990.9	1186.9	1561.4	2006.1	1838
	264		1089.9		538.3	758.6	1340.4	1171.9	933.1
	290		565.1		601.8	485.3	597.5	511.7	852.7
	294		2574.6		1541.3	3862.3	2639.9	3968.8	3955.7
	296		1821.8		1115.3	1015.1	1379.4	1444.1	867.3
	342		621		757.4	791.6	504.5	671.6	1320.3

	13a8
		Signal
	SEQ ID		EA02014_4039 0_H133A_959T		EA02014_403 91 _H133A_96 0TT	EA02014_40 392_H133A_ 961TT	EA02014_403 93_H133A_96 2TT	ThyPapCan_0201 4_40090_H133A_829TT	ThyPapCan_02014_ 40091_H133A_830TT@IC
	2		1817.9		1937.6	2632.3	2613.7	2167.7	1372.7
	3		605.6		546.8	697.5	480.4	1793.5	1236.1
	5		8452.7		11532.9	5852.5	5194.8	3735.8	2822.7
	6		2508.7		1035.1	1581.2	1561.5	1568.4	1723.3
	9		1557.3		17159 3	4950 2	1272.3	3597.7	3243.8
	12		84236		3364.4	3626 3	1198.7	59384	3878 6
	18		9560.7		5478 7	8997.6	4601 2	6673 3	6322.9
	22		57452		85846	5849.4	2275 2	4413.3	2539 9
	25		2003.7		619.6	797.7	887.6	2860.8	3257.2
	28		1280.7		1023.3	810.5	604.5	703.2	592.2
	32		5819 5		1430	6760 1	736.3	5057 7	6877 1
	39		844.6		1193.7	2035	1643.1	906.4	977.6
	74		1794.4		871.9	1342.7	1207.3	873.7	438.1
	78		1290.5		705.2	582.1	644.6	514.2	463
	143		3606.5		1206.5	2610	3369.8	2462.6	1861.9
	174		5367.9		3197	2890.4	2641.3	4481.7	4198.6
	175		6112		1232.1	3258.2	5085.3	3246.9	2442.8
	191		628.6		681.7	1024.6	538.5	913.3	723.7
	212		762		850.9	1201	690.9	797.9	784
	222		3380.6		2257.2	1397.9	127.7	892.4	646.7
	233		1273 2		3459 2	5807 2	1846 1	4170.6	4565 3
	234		4522.8		4556.7	4306.9	2687.8	3024.6	3006.1
	237		1813.8		2740.9	2387.6	1999.7	1879.2	1708
	238		1451		897.7	1411.1	1416.3	966.6	803.6
	245		841.7		1542	14342	1628 4	1415.8	1521.5
	250		834.9		839.6	1111.7	2545.8	1268.4	993.6
	252		1612		1068.9	1122.2	1770.9	1379.1	1201.1
	264		1720.7		1090.3	766.9	838.8	728.4	1170.1
	290		548.7		268.4	292.1	366.4	284.9	221.6
	294		4894.2		2878.6	1768.4	2181.6	2628.2	1888.8
	296		1074.9		850.9	729.8	975.7	1061.8	946.4
	342		1199.4		802.1	874.5	1428.7	842.7	661
	13a9

		ThyPapCan_02014_.... Signal
	SEQ ID		40092_H133A_83 1TT	40093_H	133A_832TT	40095_H133A_834TT	40096_H133A_835TT	40097_H133A _836TT	40098_H133A_83 7TT@I2
	2		2960.8		3724.8	3502.5	2185.5	2399.1	2900.3
	3		1034.7		1002.2	539.6	891.3	452.9	1885.7
	5		1648.6		2929.1	3651.5	1611.8	3731.1	3572.5
	6		1182.8		1644.7	1603.7	966.3	2282.6	1818.1
	9		1637.6		5720.9	3045.3	1451	5197.6	7768 2
	12		4300 1		3654 1	3555 9	3946.6	2299	2877.5
	18		6335 8		3922 6	35796	3282 2	3591 8	9567
	22		3973		4533	5478 5	2546.7	2145 4	7779 3
	25		7110.3		2680	2117.2	5212.6	869.3	8325.2
	28		550.3		1205.7	660.4	730.8	642.1	734.4
	32		3476 1		8075 3	2580	2524 9	2454 3	7864 8
	39		973.9		1322.9	1283.6	847.1	1328.7	1793.3
	74		611.8		1044.9	1510.9	831.5	519.6	513.4
	78		179.8		404.5	344.8	133.7	582.7	482.5
	143		2333.5		2803.8	3730.7	2073.1	2033.9	3437.3
	174		2628.9		5254.6	2333.9	922.6	5487.5	2544
	175		3237.9		5586.3	5010.7	2460.9	2800.9	4480.6
	191		3519.9		1826.9	1761.9	1360.1	531.4	2426.7
	212		957		1085.3	1218.4	764.9	1307.9	1161.5
	222		3373.3		1016	723.7	3010.1	829.7	1642.5
	233		11130 3		16096 3	16100 3	10534.6	2873 8	13024 1
	234		2382.4		2253.4	2236.6	842.4	1767	3577
	237		2141.4		2712.7	2749.7	2120.8	2269.8	2385.2
	238		719.8		2489.2	1179.1	806.1	1516.4	1336.1
	245		873 3		1325.6	1693	870.8	1916 1	1869.5
	250		2074.6		1295	2234.8	1654.7	2588.9	1104.7
	252		1496.7		2444.6	2031.7	1605.8	1808.1	1897.2
	264		931.2		1035.7	1188	746.7	1074.1	1170.2
	290		499.8		667.3	663.2	626.9	634.4	625.8
	294		2010		4135.1	2098.4	1207.1	3722.1	2055.4
	296		2128.8		2051.6	2507.5	2156.5	1001	2034.3
	342		835.3		1453.2	1071.6	615.5	1084	1209

	13a10
		Signal
	SEQ ID		ThyPapCan_02014_40 099_H133A_838TT		02014_40317@I2_H133A_839TT	pc_EA40200 _VDX879TT	pc_EA40201 _VDX881TT	pc_EA40202 _VDX882TT	pc_EA40203_ VDX883TT
	2		3540.3		1657	3331	3253.1	3349.3	3642
	3		1796.3		750.6	1334.9	1248.1	477.8	697.9
	5		4954		2602	3011.2	2106.6	1504.9	2070.9
	6		2558.4		1246.9	1008.5	1708.8	874.6	704.3
	9		4538		3328.9	1097 2	1789 6	857 2	1327.3
	12		2544		64206	5055.9	4641.7	3423 2	2283
	18		9287.1		5085	6074 5	5157.6	7305 1	5996 2
	22		5415 4		3850	3537	3100 8	25884	2125 9
	25		12197.9		1296	3368.6	4941.8	1669.9	1166.3
	28		915.5		631	842.7	869.9	606.5	459.6
	32		4804 9		3883.4	3039.3	5722.3	3401.7	1964.8
	39		1409.2		756	1855	1356	1421	1878.4
	74		992.8		349.3	597.9	708.5	741.6	421.6
	78		352		259.9	342.4	434.8	172.4	207.2
	143		4199.6		1314.8	2573.1	5070.9	4302.9	3455.3
	174		3924.5		1399.1	5622.6	4217.6	1231.4	1538.6
	175		6697.7		1590	4211.2	7931.3	7786.3	5294.6
	191		3745.9		816.2	4875.5	3853.9	2370.5	1807.8
	212		1219.1		1064.8	1480.3	1571.7	1350.2	874.7
	222		8593.9		434.6	1994.5	2235.1	980.8	748.4
	233		20899.6		4657	37907 7	22959 2	12092.7	30504 2
	234		3763.9		1496.4	3667.8	2431.2	1077.6	1447.8
	237		2711.1		2046.9	1892.2	2919	2374.3	2011.2
	238		994.1		1047.3	1092.4	929.6	877.7	776.8
	245		1339 1		1019 4	2826 7	1538 1	1379 8	1254.9
	250		1736		710.8	2570.5	1597.7	1899	1923.8
	252		2933.1		1316.3	1849.2	2395.5	2621	1992.7
	264		1426.6		987	2826.4	1272.7	2511.5	1495.4
	290		666.3		368.1	487	665.4	692.1	706
	294		3175.8		1237.3	4622.7	3449.7	976.8	1074.7
	296		1837.4		899.1	2639	2217.5	2398.4	2049.3
	342		1355.2		406.1	1662.9	803.3	1501.3	863

	13a11
		Signal
	SEQ ID		pc_EA40204_VD X884TT	pc_EA40205_ VDX885TT	DX886TT	pc_EA40206_V	pc_EA40207_ VDX890TT	pc_EA40208 _VDX892TT	pc_EA40209_ VDX893TT
	2		2895.1		2994.7	3397.6	4328.1	4435.3	5980.3
	3		1144		932.3	1277.6	3294.1	1361.2	475.7
	5		1096.3		1151.4	4943.4	1741.8	3416.4	653.5
	6		916.1		945	1696.2	1593.6	1278.6	740.7
	9		2396 9		3699 9	1671.3	1488.6	2591 9	1644 7
	12		3762 2		3308 4	3095.3	2865 4	5996.8	1644.2
	18		9468 3		6669 2	10475.1	18044 8	9220 2	11224.4
	22		3823 4		28134	4326 9	7811.1	7837	1650 1
	25		1322.8		496	4227.5	4437.5	1271.9	878.1
	28		563.6		333.7	602.3	782.9	724.2	636.5
	32		4985.4		854 7	1788.8	24236	2099.4	629.4
	39		963		1562	1937.7	1973.7	1774.1	1387.6
	74		417		443.9	383.6	600.6	814.2	578.6
	78		169.9		266.2	346.4	207.7	507.4	117.7
	143		2357.9		2936.4	2961	6472.6	4513.4	3395.7
	174		1096.5		2227.9	1825	1268.5	5715	380.6
	175		3559.1		4964.6	5395	12711.3	7961.8	5835.7
	191		5320.2		8526.6	4668.1	4129.5	8005	4597.4
	212		1168.2		1634.1	1396.4	1197.9	1161.5	1523.4
	222		805.9		739.2	2330.2	3890.4	873.8	1137.4
	233		25920.6		37919 3	33438	7762.1	23325.9	20940.7
	234		1882.8		1513.3	2438.5	4528.9	2232.7	1253.9
	237		2950.6		1089.9	1886.5	2930.8	2484.4	2082.4
	238		1037.6		1240	818.8	709.9	1096.2	877.7
	245		1151 5		1872 7	2007.7	1316.8	2042 3	17174
	250		3324		2153.4	2363.7	1885.4	2787.9	3192.2
	252		1641		1236.4	1558.6	2605.7	1944.3	2613.4
	264		1120.1		2994.2	1776.5	1401.3	1517.1	2362.8
	290		624.5		516.4	692.1	520.9	778.6	1209.7
	294		1273.1		3050.2	1557	1442.8	3564.9	424.1
	296		2601.3		2023.6	2244.2	2054.1	2777.3	4347
	342		723.2		973.5	587	2028.3	679.8	746.2

	13a12
		Signal
	SEQ ID		pc_EA40210_ VDX894TT		02014_40319_H 133A_903TT	02014_40320_ H133A_904TT	02014_40328_ H133A_928TT	02014_40330_H 133A_932TT	02014_40331_ H133A_933TT
	2		4563.8		3729.2	2859.9	2475.3	3977.2	3386.3
	3		475.3		827.4	4626.7	246.3	2009.2	1481.9
	5		1717.5		3130.1	1505.9	1737.4	1875.4	4763.8
	6		698.9		1153.2	2668.3	1104	1295.6	1385.4
	9		3017.4		3184.7	63162	1911 3	3801.9	2397 6
	12		1662.3		4850	7880 2	3035	58742	3637 2
	18		7186		6997.2	14099	11077 7	97246	10100 8
	22		1195.2		35834	60582	4793 7	6322 5	4421.3
	25		587.7		868.9	28372.6	667.2	3526	4180.2
	28		728.8		621.5	1685.2	927.3	646.4	882.8
	32		1503.2		2517.7	19668 7	3607 7	1986 9	6399.2
	39		1609.6		1035.8	1140.8	1205	1028.3	1359.6
	74		240.3		718.9	623.5	583.2	953	1021.1
	78		196.7		180.1	370.1	543	465.1	373.7
	143		1838.3		3062.8	1513.2	2903.2	3977.3	3056.1
	174		1548.4		1810.3	1087.6	2910.3	3511.6	3939.4
	175		2462.7		5545.8	1615.4	4518.2	7011.2	5184.6
	191		3292.3		2497.1	3479.2	1085	5805.4	2286.3
	212		1289.5		1230.1	645.1	1795.6	639	1130.8
	222		502.2		315.3	8484.1	1685.8	4996.9	611.8
	233		40786.7		16562	17697.8	3636 9	19787 1	4648.9
	234		1248.4		1443.8	3706.7	2565.5	2733.9	2463.9
	237		1286.1		2852.7	2832.7	2610.7	2178.6	2681.6
	238		1382.7		1243.2	990.6	1636.4	1423.2	1469.2
	245		2174		1000 7	703.1	2029 7	1000.1	1302.2
	250		2738.6		2792.3	1076.8	1439.3	3692.8	2113.9
	252		1078.4		1615.7	2091.1	2366.6	1895.9	2343.2
	264		2550.8		948.7	659.3	1351	811.7	1104.5
	290		518.9		745.9	1057.1	320.1	924.2	1016.3
	294		1936.2		1348.3	757.5	2287.3	2252.8	2351.2
	296		2194.3		1614.6	1446.4	729.6	2955.6	1690.2
	342		612.9		748.4	366.7	1061	1424.5	894.4

	13a13
		Signal
	SEQ ID		EA02014_403 75_H133A_92 2TT		EA02014_40381 _H133A_945TT	EA02014_40382 _H133A_946TT	EA02014_40383_H133A_94 7TT	EA02014_4038 4_H133A_948TT	ThyFolCan_02014_40084_H133A_823TT
	2		1365.7		2250.1	3658.1	2299	3252.8	3923.8
	3		935.6		1124.4	1023.9	2053.6	1187.9	1004.7
	5		7421.5		656.7	3000.3	4151.2	1730.9	3450.3
	6		1026.6		982.6	1628.3	1939.7	1023.9	1961.6
	9		10992.7		1452.2	2478.2	4581.5	15676	3548 1
	12		3007		2413.7	5402.5	7248.9	43826	6659 3
	18		4106.5		1506 8	9107 3	6629 5	9741 9	7072 7
	22		4766 3		5493.6	3034.7	4839.3	4984.8	4552
	25		900.4		10351.1	1252.1	10112.4	3841.3	7385.3
	28		685.8		1022.4	714.8	1191.6	656.5	625.4
	32		3099		1984	3215.8	12202 3	3643.5	4762.1
	39		1012.3		480.5	1173	1094.8	781.3	1099.1
	74		987.5		61.1	894.2	877.1	629	887.2
	78		418.8		111.9	529.1	188.7	371.9	650.2
	143		2030.1		1411.5	3847.8	2492.6	2091.2	2930.6
	174		2288.4		269.9	4741.5	2231.5	2488.1	7050.3
	175		2412.3		2138.9	6427.7	3285.3	3691.4	4648.5
	191		617.2		983.6	2182.4	2820.3	2081	1043.3
	212		1018.3		208.9	1054.6	964.8	771.1	591.5
	222		725.6		8103.4	443.9	4302.9	1433	10512.7
	233		4450.9		1177.3	20110.2	19683 9	4061 3	2305 1
	234		1926		2079.3	2095.5	2384.8	2482.7	6283.2
	237		2547.1		2912.4	3040.2	2736.1	3005.4	2681.3
	238		1822.6		264.6	1023.9	929.2	1088.1	1176.9
	245		2699 8		113 4	1230.8	1359 8	887	10676
	250		1198.1		249	1802.6	1764.8	2220.8	1112.4
	252		1311.3		613.6	2434.6	2260.9	1332.7	1526.7
	264		1364.1		382.4	1206.6	1095.1	1210.5	1451.3
	290		463.8		1223.5	805.1	865.9	867.2	932.7
	294		2024.9		340.9	4085.3	1859.8	2226.1	3796.8
	296		1214		407.7	1761.4	1917.2	3476.1	1298.6
	342		619		828.4	1154.4	639.7	934.8	1110

	13a14
						Signal
	SEQ ID	ThyFolCan_02014_ 40085_H133A_824 TT		ThyFolCan_0201 4_40086_H133A _825TT		ThyFolCan_0201 4_40088_H133A_ _827TT	ThyFolCan_0201440089_H133A_8 28TT	02014_4031 8_H133A_84 0TT	fc_EA40212_VDX896TT
	2		4103.9		1719.4	2824.5	3008.4	2542.7	2391.5
	3		2480.6		144.5	880.5	497.6	1222.4	1069.4
	5		2190.2		1199.7	4743.6	2936.8	3696.7	776.8
	6		678.7		2589.2	2824.2	1605.5	1411.7	1403.4
	9		6166.7		326.1	5428 5	2131 9	9187.9	10000.2
	12		2872 9		12574	4087 8	3981.7	7045.4	3506 5
	18		2329.3		4765.3	11522.2	72662	30454	4683.8
	22		4848.8		1224 5	3611.1	6001.8	8784 3	1719.5
	25		11319.1		234.5	3308.3	557.1	3685.8	383.8
	28		772.7		206.8	830.6	1057.1	1256.5	1118.8
	32		2272		1393 6	5410.1	3330.7	13825	869
	39		892.8		541.9	838.5	941.3	1041	3461.7
	74		126.8		2071.9	988.3	1357.4	696.8	1290.4
	78		253.6		307.6	635.6	920.6	422.6	705.5
	143		2718.2		1570.7	2322.8	2621.6	1038.8	4569.2
	174		323		1399.8	2817	9345.5	876	8849.8
	175		4437.4		1584	4411.6	4784.9	1387.4	7804.9
	191		2214.1		575.3	641	1208.4	1370.9	4609.8
	212		1667.2		662.2	577.7	1864	1229	590.6
	222		13107.5		470.6	1030.1	3020.4	431.9	641.9
	233		3196		1087 3	2932.3	7081	12626 7	4567 8
	234		1659.8		875	2243.3	1839.4	1328	7454.4
	237		4693.9		830.4	3700.2	2563.5	3040.5	1008.9
	238		206.3		244.8	1695.9	2546.2	1614.2	1880.4
	245		248 7		425	1273.9	1215.4	1333 6	2807 1
	250		935.9		379.8	2284.4	894.4	867.2	844.3
	252		1005.5		1023.3	1166.6	1884.3	1524.6	678.4
	264		231.5		589	1480.9	1740.6	670.9	2459.5
	290		638.4		233	961.8	668.6	430.5	996
	294		253.4		1349.8	3087.1	7034.2	1137.3	7044.6
	296		1530.8		1570.6	1591.5	1110.6	1647.7	1498.9
	342		1457.8		712.8	1052.1	946.9	436.8	1893.8

	13a15
		Signal
	SEQ ID	fc_EA40214_ VDX898TT		fc_EA40215_VDX899TT	X900TT	fc_EA40216_VD	fc_EA40217 VDX901TT	_fc_EA40218_ VDX902TT	fc_EA40221_ VDX909TT
	2		2470.7		3328.2	4429.9	993.8	2649.9	4427.4
	3		226.4		523.3	1216.2	230.6	417	2364.4
	5		1037.2		3614.5	1440.6	3319	1487.4	9590
	6		997		685.7	1641.1	967.8	2453.4	2537.2
	9		384 9		1897 9	973.8	468.7	915.8	3339 3
	12		9940.9		1131.5	4352 5	3202.2	4269.6	15203.8
	18		4150 7		7715 2	5736.9	555.2	9139 4	7575.1
	22		499.6		4466	2120 9	208.2	1563 8	1471.9
	25		1721.2		1912	10485.2	826.1	1002	6640.8
	28		232.1		706.8	501.4	122.6	779.4	645.5
	32		2728		3753	2644 6	1820.8	3404 5	10045 3
	39		1006.6		1103.3	1192.6	880.8	2310.8	346.3
	74		2642.5		794.9	374.8	1621.8	1057.9	294
	78		294.6		280.8	131.2	136.4	344.4	391.3
	143		2141.4		2616.6	5902.7	3882.4	3860.3	2140.3
	174		407.1		2263.7	4270	2006.5	2076.1	4365.1
	175		2416.2		3697.7	9897.3	6489.5	5706.6	2832.4
	191		1436.8		2900.8	5153.2	165.9	963.5	512.1
	212		839.5		1241.8	173.4	186.6	451.1	539.9
	222		293.5		1318.1	6645.4	426.3	623.3	2727.5
	233		17314 7		18164.1	2971 6	2885.8	2068 7	15099.5
	234		2834.4		1575.6	3547	904.6	3783.5	6440.1
	237		945.9		3105.6	1268.9	2192.9	893.1	2333.7
	238		702.9		1189	744.7	554.4	383.3	908.6
	245		9146		1729.7	1038.5	8394	7842	459 1
	250		845.9		2673.5	2175.8	702.9	1305.7	723.2
	252		1300.4		2454.3	1377.7	1061.4	1235.8	2869.5
	264		467.6		1277.4	638.7	2058	1076.7	569.3
	290		573.7		617.2	993.4	767.4	1281.3	866.1
	294		507.1		1922.1	2827.1	1753.3	1585.8	2926.6
	296		2156.2		2563	1832.8	533.7	2130.3	2647.8
	342		535.6		937.4	2261.2	1949.2	975.7	618.5

	13a16
		Signal
	SEQ ID	fc_EA40222 _VDX910TT		EA02014_40386_H133A_954TT		EA02014_40394 _H133A_967TT	EA02014_40395 _H133A_968TT	EA02014_40396_H133A_969TT	EA02014_40397 _H133A_970TT
	2		2362.1		1417	1834.4	2135.5	3006.2	1378.8
	3		254.5		263.8	293.1	346.3	346.5	230.2
	5		2789.1		8005.6	1358.8	637.3	1214.1	1214.7
	6		1096.6		1050.7	1291.7	987.4	1223.8	2564.6
	9		834 8		1052.4	2701.2	1171.4	6080	4404
	12		2031.6		4283 7	2805 1	4294.1	4966.4	1686 4
	18		9498.5		18893.2	15454.8	23854	3575 2	14192 8
22			1449.5	1020	888 3	25854		3721.5	727 2
25			541.3	253.5	572.5	3378.9		709.2	372
28			502.3	443	868.6	378.9		964.1	341.9
32			1205 9	7122 3	811 1	2553 9		797	1753 4
39			1215.4	1031.1	511	722		2166.1	527.2
74			1385.4	1477.5	509.1	1159.3		1031.4	495.4
78			367.5	834.2	200.7	235.3		433.7	262.9
143			1956.7	1083.1	1486.7	2746		2538.4	1943.7
174			4466.4	2823.7	1156.3	3547.9		1541.5	2214.3
175			1799.1	1264	1758.7	4539.5		3663.3	2264.4
191			560.2	1494	607.9	2943.2		2727.4	1474.7
212			1046.1	307.8	548.8	340.4		982.9	647
222			248.3	533.1	1356.4	1194.2		1169.8	228.2
233			1183 6	612 7	1063.5	5528 1		6265.3	1076
234			1886.7	3460.3	1207.2	2384.1		3860.6	3322.7
237			934.6	933.5	2133.3	791		1890.7	1257.5
238			834.9	291.1	1034.1	380.8		946.4	415
245			1509 7	786	657.1	384 9		47734	447.6
250			2406.8	1245.7	1247.1	659		1647.1	595
252			1536.8	1182.4	1035.6	526.7		893	1276.7
264			1315.8	582.8	1373.3	958		1622.3	508.6
290			732.4	835.3	683.8	1744.3		523	850.2
294			2817	2372.1	929.5	2611.8		1279.4	2097.3
296			1088.4	1541	2014.6	621.4		1368.3	981.8
342			763.6	866.9	722.2	559		594.3	445.9

13a17
	Signal
SEQ ID			EA02014_40405_H133A_982TT	EA02014_40406_ H133A_983TT	pb1	pb2	pb3	pb4	pb5'
2			3615.6	2106	204.1	324.2	178.6	206.6	249.1
3			765	372.2	171.5	303.8	159.1	61.1	90.2
5			2446.1	394	107.9	19.9	68.8	74.6	69
6			1051.6	1176	211.6	214.4	318.6	218.8	355.4
9			2059.2	1081.4	18	18.8	29 2	9.7	134
12			3520	391 5	11.4	9.1	9 9	33.8	8.3
18			46334	13339	942	50 7	67 6	74.9	29 7
22			6678 8	403.5	80.1	8.8	58 9	6.8	5
25			1229.9	601.5	197.1	106.7	65.2	18.7	87.1
28			778.2	197.6	11.4	9	6.6	11.3	1.7
32			1302 9	4182.6	8 7	216	30 5	66 1	646
39			2104	699	246.6	121.1	42.8	122.8	171.7
74			1189.7	1142.3	59.9	94.4	70.3	29	5.2
78			833.6	80.9	48	45.5	66.9	49.5	119.7
143			2842.2	1778.2	290	291.2	190.4	65.6	203.5
174			3307.3	329.8	29.8	42.8	25.8	11.5	13.9
175			3878.9	1966.8	165.7	282.2	158.3	67.2	249.2
191			3952.2	5278	46.8	181.4	148.2	97.3	152.1
212			2201.7	1134.3	70.4	55.7	3.5	87	99.8
222			469	451.7	120.4	148.4	57.7	49.6	58
233			11069 2	5607	13	9.8	76.9	7.6	5.6
234			3590	3449.5	186	201.3	165.3	54.8	141.1
237			2057.6	740.2	135.3	150.7	16.6	14.5	19
238			1679.4	432.3	22.5	43.8	11.4	6.2	5.1
245	2301.6		1040.5	7.1		12.7	22.5	5.9	4.6
250	434.6		1079.4	97.8		138.9	33.7	71.3	46.8
252	844.6		1446.1	35.6		80.3	29.6	63.3	88.5
264	1125.7		1084.5	102.8		31.6	147.6	101.5	163.3
290	347.9		466.9	35.8		7.3	14.4	87.2	71.7
294	1601.7		575.2	105.1		94.5	41.5	31.9	35.3
296	872.8		1349.1	217.8		162.6	186.2	171.2	212.5
342	948.7		482.8	296.5		220.2	308.1	126.3	298.6

13a18
	Signal
SEQ ID	pb6'	pb7'	pb8'	pd10	pd11	pd12	pd9
2	167.6	233.9	232.6	116.9	204.5	293.2	79.6
3	110.3	88	221	90.7	74.9	90.9	129.5
5	29.7	70.2	182.5	32.6	34.5	130.5	12.9
6	226.5	440.1	207	219.7	275.2	134.1	193.1
9	28.9	26.8	30.9	32.4	5.3	25.7	20.3
12	6.6	18.8	61.4	38.2	10.7	4.1	11.6
18	53.6	37.8	31.8	68.8	117.5	70.8	23.4
22	16.9	10.6	32.1	6.4	29.2	21.2	3.1
25	127	123.7	118.7	9.7	10.6	64.9	6.6
28	13.2	4.9	43.3	8	8.1	12	26.6
32	36.4	51.5	25.1	17.2	17.2	19.7	11
39	326.5	182.6	27.3	126.2	151.9	26	182.7
74	48.2	29	39.8	55.3	35.3	76.9	6.2
78	92.1	20	93.1	26.9	11.9	56.6	104.8
143	176.4	224.5	248.5	126.2	184.6	162.8	249.6
174	40.8	149.8	24.2	10.6	15.6	81.4	16.8
175	262.5	355.4	207.8	21.6	213.9	175.8	193.7
191	151.2	310.7	93.3	65.7	108.4	95.9	79.3
212	93.6	121.7	141.7	30.8	42.6	108.4	93.2
222	83.5	82.8	60.7	16.4	50	71.1	66.9
233	144.7	64.1	16.5	7	6.4	70.8	8.1
234	40.4	248.1	43.8	45.5	143.3	30.9	77.2
237	223.8	141.1	129.5	57.6	21.9	11.5	18.7
238	32.1	27.6	66.5	10.5	33.4	7.7	61.6
245	19.8	7.2	4.8	2.4	11.2	4.7	6.7
250	75.3	109.2	65.3	85.7	119.9	118.7	66.4
252	103.8	91.2	94.8	17	69.3	76.1	22.5
264	151	224.6	129.9	122.8	99.8	61.5	24.8
290	21.8	27.6	50.7	49.3	6.2	2.7	3
294	91.1	58.4	40	24.8	52.6	67.8	31.7
296	282.4	299.2	143.7	149.7	197.9	205.2	147.7
342	304.5	437.8	327.4	90.9	247.6	224.4	268.6

Table 13b

		13b1
			ThyMixBen_02014_....._Signal
		SEQ ID	40062_H133A_ 800TT	40063_H133A _801TT	40064_H133 A_802TT	40065_H133 A_804TT	40066_H133A_ 805TT	40067_H13 3A_806TT	40068_H13 3A_807TT
		83	156.7	114.3	73.8	240.3	173	193.6	291.2
		142	28.3	7.9	15	28.4	26.9	25.8	14.2
		136	100.4	23.4	15.3	106.9	28.3	14.9	56
		137	527.2	47	32.3	177.2	19.5	53.1	404.6
		152	237 5	230 4	188 7	465 2	183	260 9	190 9
		160	291.6	141.9	80.9	323.1	98.1	120.3	219.7
		163	15.6	53.1	25.1	70.8	20.1	56.1	47.8
		165	190.2	230.7	142.4	483.5	179.3	241.7	410.9
		210	436.9	8.6	18 4	343 7	31 8	8	148 1
		214	269.2	134.4	167.7	141.8	145.7	140.5	349.1
		219	217	19.6	7.9	85.6	21.8	26 7	115 1
		224	588.2	121.4	119.2	275.2	136.6	240.5	392.5
		225	508.1	30.1	42.3	107.3	59.4	29.5	147.3
		226	57.3	48.2	80.9	103.9	43.5	12.3	88.9
		309	257 7	20 6	20 7	75 5	19 2	24	833 9
		332	287.1	17	26.1	103.8	115.9	68.8	37.9
		346	622.2	18.3	24	17.6	20.7	13.2	442.1

		13b2
			02014_...._Signal
		SEQ ID	40198_H13 3A_871TT	40321_H13 3A_913TT	40322_H133A_914TT	40323_H133 A_915TT	40324_H133A_917TT	40325_H133A_9 18TT	40326_H133A_919TT
		83	39.5	90.2	76.4	255.8	150.8	109.7	142.6
		142	253	225	129	34.4	85	206	67 7
		136	9 8	31 8	16 8	132	115.4	130	7 2
		137	227.8	53.6	68.7	60.1	29.6	133.2	29
		152	1844	214.3	253.2	294.7	313 8	251 3	294 2
		160	267.5	250.7	206.5	328.8	191.1	220.8	114.4
		163	44.3	56.4	23.9	98.5	35.2	68	41.8
		165	174.3	221.8	185.5	266.3	104.6	217.1	159.9
		210	25.8	153.6	42	55.5	8.2	152	49.1
		214	43.2	83.6	110.6	213.7	132.6	89.3	138.7
		219	56.6	53 5	45.2	122 1	24.4	124	41 3
		224	269.7	357.4	260	191.5	168.8	330.8	169.8
		225	204.8	322.6	114.2	55.6	121.8	317.6	95.8
		226	11.9	57.4	69.9	22	67.5	33.6	19.3
		309	37 9	37	28.2	53	10 2	32.6	24.2
		332	9.9	59.2	66.7	57.8	13	40.5	9
		346	28.6	263.8	11.1	27.2	32.5	153.4	61.1

		13b3	ThyFolBen_02014_...._Signal
	SEQ ID		40079_H133A_818TT		40080_H133A_819TT		40081_H133A_8 20TT	40082_H133A_8 21TT	40083_H133A_822TT
		83	63.4		151.8		104.1	27.5	138.9
		142	15		20 2		18.8	184	23
		136	176		1354		771	317 7	177
		137	24.7		36.1		18	28.4	42.6
		152	62.1		176 7		1464	273	328 2
		160	153		138.1		131	192.4	124.1
		163	6.9		21.4		35	63.5	13.4
		165	28.7		155.3		112.6	135.2	222.6
		210	27.5		104		8 1	9.9	32 7
		214	211.7		210.8		83.9	166.4	160.2
		219	18.7		111 8		45 7	10.9	296
		224	114.7		241.8		100	160.9	242.4
225		69.4		63.8		145.4		110.9	126.1
226		92.1		95.5		36.3		74.5	67.2
309		16 8		17 1		12 1		18.8	27 3
332		40		20.5		21		11	6.6
346		7.2		27.1		18.6		29.3	26.7

13b4
	fa_EA40...._Signal
SEQ ID	191_VDX 862TT	192_VDX 863TT	193_VDX 864TT	194_VDX 865TT	195_VDX 866TT	196_VDX 867TT	197_VDX 869TT	219_VDX 907TT	220_VDX 908TT
83	26.9	24.4	56.7	155.1	22.3	94.8	63	41.9	256
142	16 1	224	14 5	54.8	17.1	15 8	15	21 9	23.9
136	23	59	204	121.4	12 5	884	77.1	47.1	36 8
137	26.7	32.4	19.4	45.2	32.8	30.8	55.2	97	63.4
152	75.7	189 4	211.4	320.4	194	75	314	114.8	260
160	239.5	101.4	91.3	292.1	55.8	86.8	53.7	47.4	145.4
163	48.5	47.6	23.1	168.3	35.4	13.4	51.5	13.5	60.4
165	26.5	94.3	81.1	197.9	112.9	164.6	155.9	29.7	250.4
210	32	162	23.8	81 4	25 3	22 7	140.5	15 5	200.8
214	119.9	130.9	141.7	235.4	263.7	30.4	124.9	57	241
219	80.6	7.6	15.9	20.9	25	10	16.5	23 4	20.1
224	50.9	25.8	50	80	32.6	99.7	136.4	137.5	167
225	66.7	41.4	64.4	69.5	18.1	94.7	51.1	51.6	149.8
226	76	2.8	3.5	99.1	88.6	18.3	60.6	6.4	11.6
309	6.7	15 2	9 4	62.6	20.2	18.4	15.9	10.2	33.3
332	9.6	36.9	10.8	91.1	103	30.1	12.3	19.7	36.5
346	25.1	16.5	20.8	89.5	34.2	20.9	18.3	11.3	114.6

13b5
	02014_...._Signal
SEQ ID	40327_H133A_920TT		40332_H133A_938TT			40334_H133A_941TT		40335_H133A_940TT
83	216.8		145.1			58.1		102.1
142	222		79			66		14.5
136	717		30.1			47 9		57 1
137	47		48.1			69.1		57.4
152	252 7		189			184 9		138 7
160	153.6		179.9			121.9		66.6
163	53.3		13			69.1		38.6
165	146.9		146.7			85.1		120
210	24		59 1			21 5		23.8
214	90.5		224.7			178.7		139.3
219	364		175			14.9		633
224	180.2		126.6			154.2		161
225	59.7		197.5			83.8		115.7
226	31.9		58.1			33.7		34.5
309	27.1		7.7			62		15.6
332	83.8		7.3			40.7		47.6
346	17		18.8			27.6		24.4

13b6
	EA02014_...._Signal
SEQ ID	40374_H133A_921TT		40376_H133A_923TT		40377_H133A_924TT		40378_H133A_925TT	40379_H133A_926TT	40380_H133A_927TT
83	174.4		32.7		69.1		86.6	67.7	204.9
142	13 7		19 8	16 5		57 7		229	189
136	24 2		93 3	121		25 3		1904	163
137	38.3		143.4	28.2		77.5		156.1	255.2
152	178 2		230 8	155		237 4		235 7	70
160	125.2		133.5	74.9		197		170.1	234
163	54.4		4.9	35.5		46.1		65.2	59.9
165	218.9		155.1	206		85.6		158.1	532.5
210	36 8		25	20 6		25 8		19 8	551 3
214	126.7		186.1	167.2		114.1		191.9	161.4
219	32 3		25 5	153		10.2		956	185
224	220.8		301.9	129.7		178.8		308.2	270.9
225	158		221.8	127.9		65.6		269.4	60.6
226	37.5		33.4	9.6		57.2		80.8	18.1
309	26.1		16	17.8		31.8		32.6	49.2
332	26.3		38.5	6.7		11.6		73.5	21.6
346	20.7		151.4	21.6		19.2		179.4	53.6

13b7
	EA02014_...._Signal
SEQ ID	40387_H133A_955TT		40388_H133A_956TT	40389_H133A _957TT	40390_H13 3A_959TT	40391 _H 133 A_960TT		40392_H13 3A_961TT	40393_H133A_962TT
83	29.1		121.5	70.7	28.8	121.6		120.5	34
142	16 7		14 2	10.8	21.4	18 6		16 5	21 9
136	15.7		27 8	67 7	45 9	15.4		8.2	43
137	28.5		44.8	34.5	50	31.3		162.2	18.5
152	85.4		163	183.4	127.1	298.3		193	221 2
160	121.9		51.9	175	137.4	208.5		125.9	162.9
163	47		27.7	43.6	80.1	29.7		33.8	44.4
165	42.2		122.9	103.8	150.6	253.3		106.9	221.6
210	27 1		5.2	20 4	73.7	29.4		38 7	85
214	36.4		68.3	165.9	25.3	129.8		165.9	100.9
219	23.3		51	29.1	25.7	67		20 8	21
224	143.8		95.1	128.8	78.7	108.8		152	109
225	140.5		83.1	34.3	54	62.8		114.7	94.3
226	4.3		24.7	84.1	47.7	33.3		66.1	13.3
309	18.7		11 4	26.7	44 2	20 6		34.1	35 2
332	11.1		4	37.9	14.1	17.8		13.1	11.5
346	21.8		22.1	12.8	35.7	38.7		29.6	10.1

13b8
	ThyPapCan_02014_...._Signal
SEQ ID	40090_H133A_829T T	40091_H133A_830T T@IC T	40092_H133A_831T T	40093_H133A_832TT	40095_H1 33A_834T	40096_H133A_835TT	40097_H133A_836T	40098_H133A_837T T@I2	40099_H133A_838TTT@I2
83	304.3	199.4	258.5	171.4	112.3	186.5	28.5	137.1	85.9
142	26	34.7	22.4	19.1	16.4	25.8	12.1	22.5	15.4
136	122	116.6	21 7	27 3	30 7	110.2	143.8	14 1	140 7
137	40.7	72	23.8	36.8	19.5	31.5	29.6	29.4	32.5
152	611 5	6826	236 2	170.5	2006	104	1364	118	155.9
160	218.1	244.7	225.4	68.9	76.4	137.3	83.2	171.7	66.7
163	17.8	119.7	38.9	51.8	52.9	42.7	25.5	57.2	23.6
165	370.6	263.3	353.8	154.1	165.1	233.2	100.4	227.9	213.3
210	223 2	249.8	303	9 7	30 2	94 5	2.1 9	42 1	23 1
214	178.4	246.7	155.4	188.3	138.6	133.7	69	298.6	90.8
219	36 8	1364	64.9	24.3	19 1	25 9	11.7	13 6	23 8
224	364.7	427.6	180	191.1	163.6	211.5	36.2	303.4	164.5
225	333.9	213.5	221	124.6	137.2	195.1	46.9	145.6	34.3
226	179.2	142	57	15.4	13.7	91.8	33.5	132.6	38
309	88 8	1366	48 8	22.1	236	40 5	16 5	33.9	24 7
332	92	56.9	42.4	61.1	34.4	70.6	14.5	108.1	60.8
346	58.4	61	197.1	38	29.1	281.5	37.5	30.8	33

13b9
Signal
SEQ ID	02014_40317_H 133A_839TT		pc-EA40200VDX879TT	pc_EA40200_VDX381TT		pc_EA40202_V DX883TT		pc_EA40203_VDX884TT	pc_EA40204_VDX884TT
83	288.1		25.7	32.9		29.2		58.6	184.4
142	161		21.2	235		7 6		21.5	306
136	62 8		160 5	141 2		8 7		13.2	194
137	45.4		155.5	35.5		63.9		41.5	31.1
152	3834		197	90.6		123.7		188	240
160	142.9		79.7	181.6		205.4		157.5	137.8
163	60.7		102.6	138.6		54.2		51.3	9.1
165	238.4		123.6	227.1		113.8		224.6	241.9
210	134.1		184.4	28 4		42.7		12.1	10
214	189.2		173	50		164.7		109.2	250.7
219	26 8		304	13		78.6		34 3	34.8
224	215.8		139.5	255.3		96.9		181.3	163.2
225	167.5		35.2	98.1		32.4		221.4	54.3
226	49.4		43.4	84.3		16.4		141.3	27.2
309	3408		33.7	32		22 8		29.8	45 9
332	15.9		39.9	67.1		14.3		88.5	25.2
346	14.3		37.9	36.5		33.5		28.7	37.4

13b10
pc_EA40....Signal
SEQ ID	205_VDX885TT	206_VDX886TT		207_VDX890TT 2		08_VDX892TT	209_VDX893TT		210_VDX894TT
83	22.9	81.5		34.1		159.2	88.7		43.6
142	26.4	22.6		8 7		10.8	18 5		23 6
136	28 1	182		96		146	17 3		22.5
137	207.7	94.4		68.8		83.7	17.8		140.4
152	77 3	225 5		227 5		95 5	301 1		59 9
160	294.5	164.6		110.3		84.5	105.6		163
163	140.9	34		25.9		9.3	41.9		83.5
165	241.8	101.4		193.6		161.9	112.8		190.6
210	416 8	24.1		45.5		15 1	26.9		15 6
214	323.3	139.5		148.2		174.1	104.5		142.1
219	131 8	12		25 9		48 9	14.4		96 2
224	215.3	136.2		126		193.8	195.4		67.1
225	49.5	105		54.2		97.4	33		38.8
226	181.1	55.2		34.1		4	15		125.1
309	89 7	474		58.6		17.8	23 5		72 3
332	73		66.8	48.2		27.5	41.1		25.3
	346		13.6	107.7	145.3	12.7	18.3	20

			13b11
02014_40...._Signal
			SEQ ID	319_H133A_903TT	320_H133A_904TT	328_H133A_928TT	330_H133A_932TT		331_H133A_933TT
			83	58.3	226.6	92.7	79.2		106.8
			142	14.5	118 3	7 4	8		13
			136	18 9	87	29 4	86.9		9 3
			137	24.4	174.5	25.7	135.9		31.8
			152	178 1	137.4	176 6	151 1		166 2
			160	83.2	205	93.5	103.6		73.3
			163	31.5	100.2	19.2	40.5		33.4
			165	223.6	235.9	161.5	132.8		132
			210	35 5	99.9	164	124 9		17
			214	118.8	178	25.6	115.6		117
			219	15.1	80.2	27 7	54 2		38.2
			224	174.5	323.7	223.3	203		185.3
			225	170.3	200.3	180.3	157.3		157.2
			226	65.2	26.9	25.2	4.6		35.7
			309	11 1	270 7	15.9	91.5		18.6
			332	12	77.4	15.1	7.2		7.3
			346	30.4	387.2	25.4	204.4		14.7

			13b12
EA02014_403...._Signal
			SEQ ID	75_H 33A_922TT	381 _H133A_945TT	382_H133A_946TT	383_H133A_947TT		384_H133A_948TT
			83	88.1	22.1	193.5	166.9		73.7
			142	12	19.6	13.2	216		16 9
			136	11.8	12.3	52	46.7		107.9
			137	51.3	182.2	43.4	23.9		14.5
			152	341	139	248.1	299.5		84.2
			160	95.6	102	99.5	184.6		90.4
			163	77.6	41.6	25.9	51.7		4.9
			165	254.8	63.5	172.6	209.2		81.1
			210	8.8	78 8	28.8	44		92 5
			214	79	94.5	148.7	101.7		82.7
			219	22.7	31 8	63.9	23.8		18 1
			224	190.5	194.7	212	278.9		201.1
			225	48.8	135.5	46.8	274.4		97.5
			226	20	24	90.8	45.3		8.8
			309	30 5	195	29 7	53.4		37 9
			332	10.4	4.2	9.2	75		44
			346	19.1	298.1	35.4	251.2		37.2

			13b13
ThyFolCan_02014_400...._Signal
			ID	84_H133A_823TT	SEQ 85_H133A_824TT 86_H133A_825TT		88_H133A_827TT 89_H133A_828TT
			83	242	70.1	71.2	24.8		145.4
			142	198	12 5	35 7	17 7		196
			136	35.7	21	594	20.3		9
			137	86.1	23.7	116.3	18.2		48.6
			152	271 9	199 6		213 1	140 9	202 5
			160	134.5	134.1		415.1	162.4	154
			163	64.5	46.1		82.1	15.5	57.8
			165	189.3	222.6		239	223.8	145.3
			210	8 8	236.3		50	21 8	33.8
			214	33.1	71.3		121.6	157.3	100.8
			219	24.8	64.8		82.1	14 1	60.2
			224	213	96.4		371.4	159.1	92.8
			225	164.9	103.3		224.4	145.2	98.3
			226	10.3	5		24	30.5	21.6
			309	1.5	33 3		38.4	14 9	19 5
			332	77.6	55.7		124.1	10.6	84.5
			346	39	124.6		31.5	29.5	21.3

		13b14
Signal
		SEQ ID	02014_40318_ H133A_840TT	fc_EA40212VDX896TT	fc_EA40214VDX898	fc_EA40214VDX899TT	_fc_EA40216 VDX900TT	_fc_EA40217VDX901TT	_fc_EA40218_VDX902TT
		83	189.9	110.7	51.4	35.8	104.6	22.8	44.7
		142	18 9	20.5	246 2	18 5	20 7	25	8 28.6
		136	22 6	1052	21 3	20 3	33.6	24	5 24 4
		137	32.3	40.6	46.3	33.8	23.9	90.6	48.5
		152	306	216.1	317.8	169.4	154 6	169	3 374.5
		160	99.5	197.5	240.4	68.7	146.7	210.5	196.7
		163	52.1	29.7	70.7	75.3	13.6	25.1	59.3
		165	236.7	142.2	147	168	174.2	28.5	89.4
		210	26.5	108.8	39.9	25.5	42.2	11	24.4
		214	115.1	63.9	59	129.9	150.1	194.1	218.2
		219	16.2	462	62.7	39.4	42.1	92.3	39.4
		224	203.9	194.6	321.6	225.2	238	212.5	187.3
		225	198	53.6	82.8	52.5	250.7	72	53.5
		226	31	57.3	111.5	66.7	24.8	41	79.9
		309	17 2	40 2	44 3	43	202	21.3	63.8
		332	51.4	26.5	129.1	7.8	5.6	37.8	31.6
		346	11	27.8	21.6	19.4	320.2	24.3	210.6

		13b15
Signal
		SEQ ID	fc_EA40 221_VDX909TT	fc_EA40 222_VDX 910TT	EA02014_40 386_H133A_954TT	EA02014_4 A_967TT	EA02014_40395 _H133A_968TT	EA02014_40396 _H133A_969TT	EA02014_40397 _H133A_970TT
		83	312.2	30.6	105.2	42.2	47.9	24.4	46.5
		142	17 8	9 9	41 7	30 5	11 8	16 7	25 7
		136	63 1	87 5	38	29.7	206.6	10.1	99 2
		137	170.4	111.7	78.6	32.1	20.5	29.4	18.2
		152	624 7	121	2154	212 7	160 9	216 7	214 3
		160	257.9	30.5	247.6	161.6	192.1	87.9	538.8
		163	21.6	27	43.5	50.7	56.9	72.4	48.7
		165	251.9	105	92.7	136.3	166.5	123.8	180.7
		210	34	37 1	49	43 9	21.1	7.4	167.3
		214	257.9	176.9	168.4	69.9	113.6	140.1	14
		219	117 7	25.9	43.2	42 1	24	12.6	57.9
		224	55.1	150	176.9	280.1	184.5	194.4	374.4
225		205.8	42.3	66.7	320.3	27	66.2	54.6
226		84.9	14.8	148.6	26	47.6	36.9	21.7
309		745.6	9.7	53.6	42	23.5	14.6	71
332		15.9	7.6	69.9	10.3	63.6	13.3	26.8
346		48	25	54.7	142.6	73.6	16.6	79.7

13b16
Signal
SEQ ID	EA02014_ A_982TT	40405_H133EA02014_40406_H133A_983TT	pb1	pb2	pb3	pb4	pb5'
83		58.1	37.8	524.7	768.1	855.6	1163.9	876.3
142		19.4	37.4	1289.3	1088.9	659.7	732.6	1415
136		94.2	11	1770.1	1622.7	523	979.3	617.3
137		17.6	111.2	1871	2554	1313.7	781.1	2087.1
152		255.8	37.6	9696.4	9404.8	1099.8	9900.7	7060
160		54.7	347.1	1273.2	1171.6	907	937.4	926.2
163		32.9	59.7	833	682.5	559.6	358.6	467.8
165		71.8	232.2	1217.8	986.3	1278.9	2537.1	1464.3
210		5.2	33.2	3401	2546.6	756.3	4893	1770.8
214		212.6	165	1384.3	1540	636.7	656.5	1213.9
219		14.1	43.6	1692.6	1911.7	912	2112.2	1417.9
224		137	228.2	2481.3	3104.2	1905.4	2292.4	2242.7
225		109.6	153.4	2367.8	2860.8	1887.6	2052.6	2116.6
226		41.6	11	797.2	969.9	617.1	626.6	920
309		20.7	30.6	5357.5	5904	3572.6	6641.9	3580.9
332		6.8	60.2	258.8	304.2	110.1	255.3	363.3
346		27	57.3	1584.2	1705.7	1507.5	1626.3	1587.1

13b17
Signal
SEQ ID	pb6'	pb7'	pb8'	pd10	pd11	pd12	pd9
83		818.3	333.4	692.1	709.8	699.7	926.2	534.7
142		1287.9	565.2	2004.2	821.9	904.8	1619.6	282.5
136		1039	962.2	1013.5	1147.9	840.9	599.8	284
137		1881.4	667.8	2058.1	2182.1	1678.9	1632.9	912.1
152		3657.7	2025	2993.5	11129.5	11824.2	11963.5	1607
160		1154.5	301.4	473.2	1849.5	1424.7	660	242
163		628.5	6534.3	1863.4	148.7	247.4	230.2	4365.5
165		1485.2	889	1904.7	1404.2	1560.4	1123.6	1919.6
210		2729.4	595.1	5286.8	7026.5	3861.8	1028.8	1724.8
214		871.1	442.3	644.5	936.1	939.1	852	347.7
219		1216.7	411.6	959.7	1085.9	1974.4	1580.2	245.9
224		2039.1	1488.3	1844.2	2280.1	2823.4	1252.3	1246.6
225		2214.8	1494.6	2186	1961.1	2628.3	1184.1	1230.7
226		822.2	437.3	684.5	540.5	839.5	1062.8	273.7
309		3787.2	1401.5	3537	3522.1	4518.1	5118.1	931.7
332		218.7	630.6	462.4	168.4	220.4	242.2	322.8
346		1682.4	909	2317.6	1193.5	1748.9	2159	622.7

Table 14 Control Genes

Control Genes for Blood
SEQ ID NO:	Gene Name

142	Bone marrow stromal cell antigen 1 (BST1)
219	Leucocyte immunoglobulin-like receptor-6b (LIR-6)
309	Bridging integrator 2 (BIN2) Control Genes for Thyroid
9	Cysteine-rich, angiogenic inducer, 61 (CYR61)
12	Selenoprotein P, plasma, 1 (SEPP1)
18	Insulin-like growth factor-binding protein 4 (IGFBP4)

Table 15a

SEQ ID	BN_800TT	BN_801TT	BN_802TT	BN_804TT	BN_805TT	BN_806TT	BN_807TT
142	28.3	7.9	15	28.4	26.9	25.8	14.2
219	217	19.6	7.9	85.6	21.8	26.7	115.1
309	257.7	20.6	20.7	75.5	19.2	24	833.9

	BN_871TT	BN_913TT	BN_914TT	BN_915TT	FA_917TT	FA_918TT	FA_919TT
142	25.3	22.5	12.9	34.4	8.5	20.6	67.7
219	56.6	53.5	45.2	122.1	24.4	124	41.3
309	37.9	37	28.2	53	10.2	32.6	24.2

	FA_818TT	FA_819TT	FA_820TT	FA_821TT	FA_822TT	FA_842TT	FA_862TT
142	15	20.2	18.8	18.4	23	10	16.1
219	18.7	111.8	45.7	10.9	29.6	28.9	80.6
309	16.8	17.1	12.1	18.8	27.3	15.2	6.7

	FA_863TT	FA_864TT	FA_865TT	FA_866TT	FA_867TT	FA_869TT	FA_907TT
142	22.4	14.5	54.8	17.1	15.8	15	21.9
219	7.6	15.9	20.9	25	10	16.5	23.4
309	15.2	9.4	62.6	20.2	18.4	15.9	10.2

	FA_908TT	FA_920TT	FA_938TT	FA_940TT	FA_941TT	Fa_EA40374 _921TT	Fa_EA40376 _923TT
142	23.9	22.2	7.9	6.6	14.5	13.7	19.8
219	20.1	36.4	17.5	14.9	63.3	32.3	25.5
309	33.3	27.1	7.7	6.2	15.6	26.1	16

	BN_EA40377 _924TT	Fa_EA40378 _925TT	Fa_EA40379 _926TT	BN_EA40380 _927TT	Fa_EA40387 _955TT	Fa_EA40388 _956TT	Fa_EA40389 _957TT
142	16.5	57.7	22.9	18.9	16.7	14.2	10.8
219	15.3	10.2	95.6	18.5	23.3	51	29.1
309	17.8	31.8	32.6	49.2	18.7	11.4	26.7

	Fa_EA40390_ 959TT	Fa_EA40391 _960TT	Fa_EA40392 _961TT	Fa_EA40393_ 962TT	PC_829TT	PC_830TT	PC_831TT
142	21.4	18.6	16.5	21.9	26	34.7	22.4
219	25.7	67	20.8	21	36.8	136.4	64.9
309	44.2	20.6	34.1	35.2	88.8	136.6	48.8

	PC_832TT	PC_834TT	PC_835TT	PC_836TT	PC_837TT	PC_838TT	PC_839TT
142	19.1	16.4	25.8	12.1	22.5	15.4	16.1
219	24.3	19.1	25.9	11.7	13.6	23.8	26.8
309	22.1	23.6	40.5	16.5	33.9	24.7	340.8

	PC_879TT	PC_881TT	PC_882TT	PC_883TT	PC_884TT	PC_885TT	PC_886TT
142	21.2	23.5	7.6	21.5	30.6	26.4	22.6
219	30.4	13.3	78.6	34.3	34.8	131.8	12
309	33.7	32	22.8	29.8	45.9	89.7	47.4

	PC_890TT	PC_892TT	PC_893TT	PC_894TT	PC_903TT	PC_904TT	PC_928TT
142	8.7	10.8	18.5	23.6	14.5	118.3	7.4
219	25.9	48.9	14.4	96.2	15.1	80.2	27.7
309	58.6	17.8	23.5	72.3	11.1	270.7	15.9

	PC_932TT	PC_933TT	Pc_EA40375 _922TT	Pc_EA40381_ 945TT	Pc_EA40382 _946TT	Pc_EA40383 _947TT	Pc_EA40384 _948TT
142	8	13	12	19.6	13.2	21.6	16.9
219	54.2	38.2	22.7	31.8	63.9	23.8	18.1
309	91.5	18.6	30.5	195	29.7	53.4	37.9
	FC_823TT	FC_824TT	FC_825TT	FC_827TT	FC_828TT	FC_840TT	FC_896TT
142	19.8	12.5	35.7	17.7	19.6	18.9	20.5
219	24.8	64.8	82.1	14.1	60.2	16.2	46.2
309	15	33.3	38.4	14.9	19.5	17.2	40.2

	FC_898TT	FC_899TT	FC_900TT	FC_901TT	FC_902TT	FC_909TT	FC_910TT
142	246.2	18.5	20.7	25.8	28.6	17.8	9.9
219	62.7	39.4	42.1	92.3	39.4	117.7	25.9
309	44.3	43	20.2	21.3	63.8	745.6	9.7

	Fc_EA40386_954TT	Fc_EA40394_967TT	Fc_EA40395 _968TT	Fc_EA40396_ 969TT	Fc_EA40397 _970TT	Fc_EA40405 _982TT	Fc_EA40406 _983TT
142	41.7	30.5	11.8	16.7	25.7	19.4	37.4
219	43.2	42.1	24	12.6	57.9	14.1	43.6
309	53.6	42	23.5	14.6	71	20.7	30.6

	pb1_Signal	pb2_Signal	pb3_Signal	pb4_Signal	pb5'_Signal	pb6'_Signal	pb7'_Signal
142	1289.3	1088.9	659.7	732.6	1415	1287.9	565.2
219	1692.6	1911.7	912	2112.2	1417.9	1216.7	411.6
309	5357.5	5904	3572.6	6641.9	3580.9	3787.2	1401.5

	pb8'_Signal	pd10_Signal	pd11_Signal	pd12_Signal	pd9-Signal
142	2004.2	821.9	904.8	1619.6	282.5
219	959.7	1085.9	1974.4	1580.2	245.9
309	3537	3522.1	4518.1	5118.1	931.7

Table 15b

Signal
SEQ ID	PC_984TT	PC_986TT	FA_987TT	PC_988TT	PC_989TT	FA_992TT	FA_993TT
142	25.1	125.8	18.5	11.4	13.7	11.8	113.6
219	17.5	25.7	22.6	11.3	32.4	61.9	25.2
309	27.6	661.8	15.7	47.4	29.8	33.2	28.4

	FA_994TT	FA_995TT	FA_996TT	FA_998TT	FA_999TT	FA_1001TT	FA_1002TT
142	20.6	25.2	34.1	21.8	31.4	12.7	74.6
219	25.9	36.3	41.5	24.9	16.4	26.5	25.5
309	16.8	26.3	19.4	22.5	14.6	32.3	47.7

	FA_1004TT	FA_1005TT	FA_1006TT	FA_1010TT	FA_1013TT	FA_1014TT	FA_1017TT
142	24.7	20.1	67.7	14.9	48.9	29.8	26.9
219	27.5	11.7	53.3	112.9	35.1	39.5	27.2
309	39.5	10.3	34.6	83.2	46.8	20	25.7

	FA_1018TT	FA_1020TT	FA_1023TT	FA_1024TT	FA_1026TT	FA_1027TT	FA_1028TT
142	25.1	25.5	35.6	26.9	27.9	15.2	24.5
219	66.3	26	6.1	26	59.3	18.8	31.7
309	31	37.4	20.6	46.9	51.4	8.8	33.3

	FA_1029TT	FA_1030TT	FA_1031TT	FA_1032TT	FA_1034TT	FA_1035TT	FC_1037TT
142	10.6	14	20	21.6	31.9	17.1	17.8
219	12.5	33.5	15.1	15	68.5	25.6	63.5
309	20.7	13	21.5	32.2	52.6	24.6	26.2

	PC_1039TT	PC_1040TT	PC_1041TT	PC_1042TT	PC_1043TT	PC_1044TT	PC_1045TT
142	16.1	22.7	99.5	129.8	20.7	79.2	16.4
219	27.9	29.5	23.6	29	21.5	19.8	38.4
309	585.3	37.6	48.7	41	32.8	59.2	112.3

	PC_1046TT	PC_1047TT	PC_1048TT	PC_1049TT	PC_1050TT	PC_1051TT	PC-FV_1052TT
142	22.4	81.2	24.7	37.5	25.9	26.6	26.4
219	11.1	97.5	61.8	14.9	14.7	109.1	30.6
309	22.5	207.8	35.4	64	39.5	56.7	28.2
	PC_1053TT	PC_1054TT	PC_1055TT	PC_1059TT	PC_1060TT	PC_1061TT	PC_1062TT
142	12.8	18.5	24.4	22.7	24.6	15.8	25.6
219	12.6	19.6	10.2	24.5	82.6	53.7	20.7
309	20.4	22.1	24	18.9	32.3	54.5	29.1

	PC-FV_1064TT	PC-FV_1065TT	PC-FV_1066TT	PC-FV_1067TT	PC-FV_1068TT	PC-FV_1069TT	PC-FV_1071TT
142	16.7	11.3	25.5	17.4	17.5	14	16.7
219	174.4	20.3	132.4	24.8	12.3	14.8	18.8
309	30.4	169.6	49.4	16.9	135.5	18.1	7.6

	PC-FV_1072TT	FA_1073TT	FA_1074TT	FA_1075TT	FA_1076TT	FC_1077TT	FC_1078TT
142	14	25.5	22.7	27	16.4	15.8	38.9
219	8.7	73.2	57	54.3	69.9	20	17.1
309	29.9	145.4	21	53	24.3	9.1	32.9
	FC_1079TT	PC-FV_1080TT	PC-FV_1081TT	FC_1082TT	pb1	pb2	pb3
142	31.9	10.2	22	19.5	1289.3	1088.9	659.7
219	40.1	17.4	38.3	88.1	1692.6	1911.7	912
309	27.1	23.4	40.8	12.9	5357.5	5904	3572.6

	pb4	pb5'	pb6'	pb7'	pb8'	pd10	pd11
142	732.6	1415	1287.9	565.2	2004.2	821.9	904.8
219	2112.2	1417.9	1216.7	411.6	959.7	1085.9	1974.4
309	6641.9	3580.9	3787.2	1401.5	3537	3522.1	4518.1

	pd12	pd9
142	1619.6	282.5
219	1580.2	245.9
309	5118.1	931.7

F. Signature Normalized to Control Genes

[0081] We further examined our 5-gene and 4-gene signatures by normalizing these genes to the three selected thyroid control genes as an algorithm for gene chip data normalization. The average fluorescent intensities of the three thyroid control genes were used for signature gene signal normalization. The performance of both signatures improved slightly when these two signatures were normalized. The sensitivity and specificity of the two signatures are listed in Table 16, and the ROC curves are shown in Figure 4a and 4b.

Table 16. The performances of the 4-gene and 5-gene signatures normalized to control genes 4-Gene Signature

	Tumor	Benign
Positive	33	11
Negative	3	27
Sensitivity	92% (0.78, 0.97)
Specificity	71% (0.55, 0.83)
5-Gene Signature
Positive	33	8
Negative	3	30
Sensitivity	92% (0.78, 0.97)
Specificity	79% (0.64, 0.89)

G. Signature Validation with Two-Round Amplified Probes

[0082] To determine if the FNA samples lack sufficient thyroid cells to provide enough probe material for hybridizing to the Affymetrix U133a gene chips after one round of amplification, two-round amplification of the target RNAs we performed two-round amplification with 47 samples that are among the 74 independent validation sample set. The data obtained show that the performances of the 5-gene and 4-gene signatures are identical with either one-round or two-round amplifications. The ROC curves of the two gene signatures with two different target preparations are shown in Figures 5a, 5b, 5c, and 5d.

Example 4

Cross validation with independent samples

A. Cross Validation with the 83 Independent Fresh Frozen Thyroid Samples

[0083] 83 independent thyroid samples were processed and profiled with the U133a chip. The number of samples in each category is list in Table 17.

Table 17. Fresh Frozen sample collection for signature validation

Sample Type	Number of Samples
Follicular Adenoma	18
Follicular Thyroid Carcinoma	1
Papillary Carcinoma, Follicular Variant	18
Papillary Thyroid Carcinoma	11
Adenomatoid Nodules	18
Adenomatoid Nodules w/Hashimoto	1
Multinodular Hyperplasia	16

[0084] The performance of the 4-gene signature and the 5-gene signature was assessed with LDA using the same cut-off value as in the training set. Both signatures gave equivalent performance in these samples, and they are comparable with the performance in the 98 training set. The sensitivity and specificity for both signatures are shown in Table 18, and the ROC curves are demonstrated in Figures 6a and 6b.

Table 18. 4-Gene and 5-gene signatures performance in 83 validation samples 4-Gene Signature

	Tumor	Benign
Positive	20	11
Negative	10	42
Sensitivity	67% (0.49, 0.81)
Specificity	79% (0.67, 0.88)

	5-Gene Signature
	Tumor	Benign
Positive	24	24
Negative	6	29
Sensitivity	80% (0.63, 0.90)
Specificity	55% (0.41, 0.67)

B. Signature Validation with the 47 Fine Needle Aspirate (FNA) Thyroid Samples

[0085] 47 thyroid FNA samples were processed and profiled with the U133a chip. The number of samples in each category is list in Table 19.

Table 19. FNA sample collection for signature validation

Sample Type	Number of Samples
Follicular Adenoma	10
Follicular Thyroid Carcinoma	2
Papillary Carcinoma, Follicular Variant	3
Papillary Thyroid Carcinoma	13
Adenomatoid Nodules	10
Adenomatoid Nodules w/Hashimoto	1
Multinodular Hyperplasia	8

[0086] The performance of the 4-gene signature and the 5-gene signature was assessed with LDA model. Both signatures gave equivalent performance in the FNA samples, and they are comparable with the performance in the 98 training set. The sensitivity and specificity for both signatures are shown in Table 20, and the ROC curves are demonstrated in Figures 7a and 7b.

Table 20. 4-Gene and 5-gene signatures performance in 47 FNA samples 4-Gene Signature

	Tumor	Benign
Positive	15	4
Negative	3	25
Sensitivity	94% (0.74, 0.99)
Specificity	62% (0.44, 0.77)

	5-Gene Signature
	Tumor	Benign
Positive	17	10
Negative	1	19
Sensitivity	94% (0.74, 0.99
Specificity	66% (0.47, 0.80)

C. Signature Performance in 28 Paired Fresh Frozen and FNA Thyroid Samples

[0087] Within the 83 fresh frozen and the 47 FNA sample collections there are 28 samples that were from the same patient. The direct comparison of our signatures in these paired samples demonstrates how well the signature will translate into the final molecular assay. The performance of the 4-gene signature and the 5-gene signature was assessed with the LDA model. Both signatures gave equivalent performance in the fresh frozen and FNA samples. These results demonstrated that our 4-gene and 5-gene signatures can perform equally well in both sample types, and proved the approach using fresh frozen samples for gene/signature identification is valid. The sensitivity and specificity for both signatures are shown in Table 21, and the ROC curves are demonstrated in Figures 8a and 8b.

Table 21. 4-Gene and 5-gene signatures performance in 28 matched fresh frozen and FNA samples

4-Gene Signature
	Fresh Frozen		FNA
	Tumor	Benign	Tumor	Benign
Positive	10	4	12	7
Negative	3	11	1	8
Sensitivity	77% (0.50 - 0.92)		92% (0.67 - 0.99)
Specificity	73% (0.48 - 0.89)		53% (0.30 - 0.75)
5-Gene Signature
	Fresh Frozen		FNA
	Tumor	Benign	Tumor	Benign
Positive	11	7	12	7
Negative	2	8	1	8
Sensitivity	85% (0.58 - 0.96)		92% (0.67 - 0.99)
Specificity	53% (0.30 - 0.75)		53% (0.30 - 0.75)

Example 5

Real-time quantitative RT-PCR

Sample Acquisition:

[0088] In order to determine whether a subset of the gene profiles and/or controls would give adequate specificity and sensitivity with RT-PCR, the following experiment was performed. The following has the advantage of requiring only one round of RNA amplification.

[0089] A total of 107 thyroid biopsies were analyzed in our study: 26 follicular adenoma, 23 follicular carcinoma, 38 papillary carcinoma, 5 normal, 3 papillary carcinoma follicular variant, 3 Hashimoto thyroiditis, 2 microfollicular adenoma, 1 diffuse goiter, 1 goiter with papillary hyperplasia, 1 Hurthle cell adenoma, 1 hyperplasia with papillary structure, 1 multinodular goiter, 1 oncocytic hyperplasia, 1 thyroiditis. Total RNA isolation was extracted by using the Trizol reagent according to the manufacturer's instructions. RNA concentrations were determined by absorbance readings at 260 nm with the Gene-Spec (Hitachi) spectrophotometer. The isolated RNA was stored in RNase-free water at -80°C until use.

Primer and Probe Design:

[0090] The primers and hydrolysis probes were designed using Oligo 6.0 and the Genebank sequences for thyroid cancer status markers (Table 22). These primers and probe sets were designed such that the annealing temperature of the primers was 62°C and the probes 5-10°C higher and amplicon size ranges from 100-150bp. Genomic DNA amplification was excluded by designing our primers around exon-intron splicing sites. Hydrolysis probes were labeled at the 5' nucleotide with FAM as the reporter dye and at 3' nucleotide with TAMRA as the quenching dye.

Table 22

Target	Accession #	Forward Primer (SEQ ID NO:)	Reverse Primer (SEQ ID NO:)	Probe Sequence (SEQ ID NO:)	Product Length
SGENE	NM_003020	gaaagcggagqagtgtcaatcca (364)	ggttttcqtctagcatcttctcttta (365)	atctacaaggacagagactggataatgttg (366)	130
TESTICAN1	AF231124	gtgagctgtgaagaggagcaggaa (367)	ctttggtcccagctcccgttca(368)	cctcaggggattttggcagtggtgggtccg (369)	102
GABRE	NM_004961	taaactccgccatcctcgtatcaa (370)	cagtggtgacaatctggcacacaa (371)	ccgtgcccatgccccgtacccatgca (372)	113
CDH3	NM_001793	ctgaagcaggatacatatgacgtgca (373)	aggatccagggcaggtttcgaca (374)	catggcagtcgcacacagtggccctqa (375)	124
FN1	NM_002026	tggtgccatgacaatggtgtgaacta (376)	catcatcgtaacacattacctcatna (377)	aagattgggagagaagtgggaccgtcaggga (378)	148
TPO-1	NM_000547	catctgtqacaacactggcctca (379)	gccacacttgtcgtcttgaggaa (380)	caaattccccgaagactttgagtcttgtgacagc (381)	148
TPO-2	NM_000547	aacctgcgtagactccgggaggc (382)	gccagtgcgtgtccacctgca (383)	ctcgggtgacttggatctccatqtcgctgg (384)	124
KCNAB1-1	NM_003471	tgaaagttccagggcttcactgaa (385)	agctgaggtagtgtgcatcccaga (386)	ctaccagtggttgaaagaaagaattgtaagtgaag (387)	138
KCNAB1-2	NM_003471	tagctgttgcgtggtgcctgagaa (388)	tgatgtcatctttgggagaacctgaa (389)	aaggtgtgagttctgtgctcctgggatcat (390)	119
FABP4-1	NM_001442	gaaagaagtaggagtgggctttgcca (391)	ggcccagtatgaaggaaatctcaata (392)	aaaggtactttcagatttaatggtgatcacatccc (393)	143
FABP4-2	NM_001442	aggaaagtcaagagcaccataacctta (394)	gacgcattccaccaccagtttatca (395)	ttgattttccatcccatttctgcacatgta (396)	123
DOI1-1	NM_000792	gggtaaatctggcccttggaactaca (397)	cccgttggtcacctagaattgagata (398)	cccagaggaagttcgtqctgttctggaaaa (399)	106
DOI1-2	NM_000792	tgcatcagatggctgggctttta (400)	gctctggttctgcatggtgtcca (401)	atgccctccccatgcatcctgcgt (402)	142
B-ACTIN	NM_001101	tcacccacactgtgcccatctacga (403)	cagcggaaccgctcattgccaatgg (404)	atgccctcccccatgccatcctgcgt (405)	295
GOLGIN67	NM_015003	gcatggtgatctttgtgaggcga (406)	ctcctggtggtcctgcatctca (407)	ctcaccaacagcgtggagcctgcgcaagga (408)	139
PAX8	BC001060	actccagcttgctgagttcccca (409)	actccagcttgctgagttcccca (410)	attattacagttccacatcaaggccgagtgca (411)	125
HERC	NM_003922	qqgtqcttttcatgaggtttgtgtca (412)	tgaggtaggcagactgtcgtaaggcc (413)	ccaacactgctgacatttctcagagatttcaaat (414)	219
DDIT3	NM_004083	cctggaaatgaagaggaagaatcaa (415)	agctctgactggaatctggagagtqa (416)	aatcttcaccactcttgaccctgcttctctgg (417)	142
ITM1	NM_152713	tggctggtcaggatatacaaggtaaa (418)	tcaacgtcctaaatgtgatgtgctca(419)	cctggataatcqaggcttgtcaaggacataaa (420)	117
C1OF24	NM_052966	tctgaaagtgataaaggaagctgcta (421)	ctttagatctgttaagctggacacac (422)	cttgaagaaacacaacttatttgaagataacatg (423)	103
MOT8	NM_018836	acggcctataacgagaccctgca (424)	gaagaggagggtcggtttccattaa (425)	ttctcacgagtgcgtcagggcatctgtgc (426)	133
ARG2	NM_00172	atttgaccctacactggctccagc (427)	gatccaatgctgatagcaaccctgta (428)	actcctgttgtcgggggactaacctatcgaga (429)	119

Real-Time Quantitative RT-PCR:

[0091] Gene specific real-time quantitative RT-PCR amplification of 21 thyroid cancer status genes and a housekeeping gene was performed using the TaqMan One-Step RT-PCR Master Mix (2x) (Applied Biosystems) and the ABI Prism 7900HT sequence detection system (Applied Biosystems). In a 25 µl one-step reaction total RNA (10ng was added to a mix that contained: 1x RT-PCR Master Mix, 0.25U/µl Multiscribe Enzyme, 0.6uM primers and 0.25µM probe. Cycling parameters were 48°C for 30min and 95°C 10min, followed by 40 cycles of 95°C 15sec and 62°C 1min. Real-time PCR monitoring was achieved by measuring fluorescent signal at the end of the annealing phase for each cycle. The number of cycles to reach the fluorescence threshold was defined as the cycle threshold (Ct value). To minimize the errors arising from the integrity of the RNA in the samples β-actin mRNA was amplified as an internal reference. External standards were prepared by 10-fold serial dilutions of known thyroid cancer positive RNA and used to ensure linearity throughout our assays. Results were expressed in mean Ct value and samples were excluded that had a standard deviation greater then one. The results are provided in Tables 23a, 23b, 23c, 24a and 24b.

[0092] The data show that the two gene signature shown in Table 23b is not as sensitive and specific as the four-gene signature from which it was derived. Table 24 shows that use of the PAX8 gene in an RT-PCR reaction as a control for thyroid-specific tissue is effective in an RT-PCR reaction.

[0093] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

Table 25

Sequence identifications
SEQ NO:	psid	Name	Accession No.	Description
1	200635_s_at		AU145351	Hs.75216 prot tyrosine phosphatase, rec. type, F
2	200771_at		NM_002293	laminin, gamma 1
3	201069_at		NM_004530	matrix metalloproteinase 2
4	201117_s_at	CPE	NM_001873	carboxypeptidase E
5	201150_s_at		NM_000362	tissue inhibitor of metalloproteinase 3
6	201185_at		NM_002775	protease, serine, 11
7	201203_s_at		Al921320	ribosome binding protein 1
8	201212_at		NM_005606	cysteine protease
9	201289_at	CYR61	NM_001554	cysteine-rich, angiogenic inducer, 61
10	201292_at		NM_001067	topoisomerase (DNA) II alpha
11	201418_s_at	SOX4	NM_003107	SRY (sex determining region Y)-box 4
12	201427_s_at	SEPP1	NM_005410	Selenoprotein P, plasma, 1
13	201430_s_at	DPYSL3	NM_001387	dihydropvrimidinase-like 3
14	201431_s_at	DPYSL3	NM_001387	dihydropyrimidinase-like 3
15	201438_at	COL6A3	NM_004369	collagen, type VI, alpha 3
16	201474_s_at	ITGA3	NM_002204	integrin, α 3 transcript variant a
17	201505_at	LAMB1	NM_002291	laminin, beta 1
18	201508_at	IGFBP4	NM_001552	Insulin-like growth factor-binding protein 4
19	201525_at	APOD	NM_001647	apolipoprotein D
20	201645_at	HXB	NM_002160	hexabrachion (tenascin C, cytotactin)
21	201650_at	KRT19	NM_002276	keratin 19
22	201667_at	GJA1	NM_000165	gap junction protein, α 1, 43kD (connexin 43)
23	201744_s_at	LUM	NM_002345	lumican
24	201792_at	AEBP1	NM_001129	AE-binding protein 1
25	201852_x_at		Al813758	Collaqen, type III, alpha 1
26	201893_x_at		AF138300	decorin variant A
27	201983_s_at		AW157070	epidermal growth factor receptor
28	202133_at		AA081084	Transcriptional co-activator w PDZ-binding motif
29	202219_at	SLC6A8	NM_005629	solute carrier family 6, member 8
30	202237_at	NNMT	NM_006169	nicotinamide N-methyltransferase
31	202286_s_at		NM_002353	tumor-associated calcium signal transducer 2
32	202291_s_at		NM_000900	matrix Gla protein
33	202310_s_at		NM_000088	proalpha 1 (I) chain of type I procollagen
34	202350_s_at	MATN2	NM_002380	matrilin 2 precursor, transcript variant 1
35	202357_s_at	BF	NM_001710	B-factor, properdin
36	202363_at		AF231124	testican-1
37	202376_at	SERPINA3	NM_001085	Ser (or Cys) proteinase inhib clade A mem 3
38	202404_s_at	COL1A2	NM_000089	collagen, type I, alpha 2
39	202440_s_at		NM_005418	suppression of tumorigenicity 5
40	202504_at	ATDC	NM_012101	ataxia-telangiectasia group D-assoc. protein
41	202575_at	CRABP2	NM_001878	cellular retinoic acid-binding protein 2
42	202588_at	AK1	NM_000476	adenylate kinase 1
43	202712_s_at	CKMT1	NM_020990	creatine kinase, mitochondrial 1
44	202796_at	KIAA1029	NM_007286	synaptopodin
45	202826_at	SPINT1	NM_003710	serine protease inhibitor, Kunitz type 1
46	202834_at	SERPINA8	NM_000029	Ser (or Cys) proteinase inhib, clade A mem 8
47	202898_at	KIAA0468	NM_014654	KIAA0468 gene product
48	202992_at	C7	NM_000587	complement component 7
49	203021_at	SLPI	NM_003064	secretory leukocyte protease inhibitor
50	203083_at	THBS2	NM_003247	thrombospondin 2
51	203180_at	ALDH1A3	NM_000693	aldehyde dehydrogenase 1 family, mem. A3
52	203228_at	PAFAH1B3	NM_002573	platelet-activating factor acetylhydrolase, isoform lb, γ sub
53	203256_at	CDH3	NM_001793	cadherin 3, type 1, P-cadherin (placental)
54	203349_s_at	ETV5	NM_004454	ets variant gene 5 (ets-related molecule)
55	203352_at	ORC4L	NM_002552	origin recognition complex, subunit 4-like
56	203354_s_at		NM_015310
57	203381_s_at		NM_000041
58	203382_s_at	APOE	NM_000041	apolipoprotein E
59	203407_at	PPL	NM_002705	periplakin
60	203417_at	MFAP2	NM_017459	microfibrillar-associated protein 2, tran var 1
61	203438_at		NM_003714	stanniocalcin 2
62	203453_at	SCNN1A	NM_001038	sodium channel, nonvoltage-gated 1 α
63	203499_at	EPHA2	NM_004431	EphA2
64	203548_s_at		NM_000237	lipoprotein lipase
65	203570_at	LOXL1	NM_005576	lysyl oxidase-like 1
66	203632_s_at	GPRC5B	NM_016235	G protein-coupled rec, fam C, group 5, mem B
67	203673_at	TG	NM_003235	thyroglobulin
68	203699_s_at		NM_013989	type II iodothyronine deiodinase
69	203700_s_at	DIO2	NM_013989	deiodinase, iodothyronine, type II, tran var 1
70	203786_s_at	TPD52L1	NM_003287	tumor protein D52-like 1
71	203851_at	IGFBP6	NM_002178	insulin-like growth factor binding protein 6
72	203854_at	IF	NM_000204	I factor (complement)
73	203859_s_at	PALM	NM_002579	paralemmin
74	203875_at		NM_003069	SWISNF related, matrix assoc, actin dep regulator of chromatin subfam a mem 1
75	203881_s_at		NM_004010	dystrophin, includes DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272 (DMD), transcript variant Dp427p2
76	203889_at	SGNE1	NM_003020	secretory granule, neuroendocrine protein 1
77	203911_at	RAP1GA1	NM_002885	RAP1, GTPase activating protein 1
78	203934_at	KDR	NM_002253	kinase insert domain receptor
79	203986_at	GENX-3414	NM_003943	genethonin 1
80	204105_s_at	NRCAM	NM_005010	neuronal cell adhesion molecule
81	204124_at	NaPi-IIb	AF146796	Na dependent phosphate transporter isoform
82	204149_s_at	GSTM4	NM_000850	glutathione S-transferase M4
83	204152_s_at		AI738965	manic fringe (Drosophila) homolog
84	204154_at	CDO1	NM_001801	cysteine dioxygenase, type I
85	204259_at	MMP7	NM_002423	matrix metalloproteinase 7 (matrilysin, uterine)
86	204260_at	CHGB	NM_001819	chromogranin B (secretogranin 1)
87	204268_at	S100A2	NM_005978	S100 calcium-binding protein A2
88	204288_s_at	ARGBP2	NM_021069	ArgAbl-interacting prot ArgBP2, trans var 2
89	204298_s_at	LOX	NM_002317	lysyl oxidase
90	204337_at		NM_005613	regulator of G-protein signalling 4
91	204416_x_at	APOC1	NM_001645	apolipoprotein C-I
92	204424_s_at		NM_018640	neuronal specific transcription factor DAT1
93	204433_s_at		NM_006038	spermatogenesis associated PD1
94	204442_x_at	LTBP4	NM_003573	latent transforming GFβ binding protein 4
95	204452_s_at		NM_003505	frizzled 1
96	204476_s_at	PC	NM_022172	pyruvate carboxylase
97	204503_at	EVPL	NM_001988	envoplakin
98	204591_at	CHL1	NM_006614	cell adhesion molecule w/ homology to homolog L1
99	204600_at	EPHB3	NM_004443	EphB3
100	204623_at	TFF3	NM_003226	trefoil factor 3 (intestinal)
101	204625_s_at		NM_000212	integrin, β 3 (platelet glycoprotein IIIa, CD61)
102	204697_s_at	CHGA	NM_001275	chromogranin A
103	204741_at	BICD1	NM_001714	Bicaudal D (Drosophila) homolog 1
104	204753_s_at	HLF	NM_002126	hepatic leukemia factor
105	204754_at	HLF	NM_002126	hepatic leukemia factor
106	204755_x_at	HLF	NM_002126	hepatic leukemia factor
107	204787_at	Z391G	NM_007268	Ig superfamily protein
108	204797_s_at	EMAPL	NM_004434	echinoderm microtubule-associated pro-like
109	204869_at		AL031664	DNA seq RP4-531H16 chrom 20p11.22-12
110	204870_s_at	PCSK2	NM_002594	proprotein convertase subtilisinkexin type 2
111	204933_s_at	TNFRSF11 B	NM_002546	TNFR superfamily, member 11b
112	204934_s_at	HPN	NM_002151	hepsin (transmembrane protease, serine 1)
113	204944_at	PTPRG	NM_002841	protein tyrosine phosphatase receptor type G
114	204964_s_at	SSPN	NM_005086	sarcospan (Kras oncogene-associated gene)
115	204975_at	EMP2	NM_001424	epithelial membrane protein 2
116	204990_s_at	ITGB4	NM_000213	integrin, beta 4
117	205051_s_at	KIT	NM_000222	v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
118	205110_s_at	FGF13	NM_004114	fibroblast qrowth factor 13
119	205153_s_at	TNFRSF5	NM_001250	TNFR superfamily, mem 5
120	205168_at	DDR2	NM_006182	discoidin domain receptor family, member 2
121	205258_at	INHBB	NM_002193	inhibin, β B (activin AB β polypeptide)
122	205286_at		NM_003222	transcription factor AP-2 gamma
123	205325_at	KIAA0273	NM_014759	KIAA0273 gene product
124	205336_at	PVALB	NM_002854	parvalbumin
125	205402_x_at	PRSS2	NM_002770	protease, serine, 2 (trypsin 2)
126	205413_at	C11ORF8	NM_001584	chromosome 11 open reading frame 8
127	205455_at	MST1R	NM_002447	macrophage stimulating 1 receptor
128	205470_s_at	KLK11	NM_006853	kallikrein 11
129	205479_s_at	PLAU	NM_002658	plasminogen activator, urokinase
130	205481_at	ADORA1	NM_000674	adenosine A1 receptor
131	205485_at	RYR1	NM_000540	ryanodine receptor 1 (skeletal)
132	205490_x_at	connexin 31	NM_024009	gap junction protein, beta 3, 31 kD
133	205531_s_at	GA	NM_013267	breast cell glutaminase
134	205593_s_at	PDE9A	NM_002606	phosphodiesterase 9A
135	205614_x_at	MST1	NM_020998	macrophage stimulating 1
136	205627_at		NM_001785	cytidine deaminase
137	205639_at		NM_001637	acyloxyacyl hydrolase
138	205683_x_at	TPSB1	NM_003294	tryptase beta 1
139	205689_at	KIAA0435	NM_014801	KIAA0435 gene product
140	205700_at	RODH	NM_003725	oxidative 3 α hydroxysteroid dehydrogenase; retinol dehydrogenase; 3-hydroxysteroid epimerase
141	205710_at	LRP2	NM_004525	low density lipoprotein-related protein 2
142	205715_at	BST1	NM_004334	bone marrow stromal cell antigen 1
143	205717_x_at		NM_002588	protocadherin gamma subfamily C, 3
144	205728_at		AL022718	DNA seq from clone 1052M9 on chrom Xq25
145	205747_at	CBLN1	NM_004352	cerebellin 1 precursor
146	205778_at	KLK7	NM_005046	kallikrein 7 (chymotryptic, stratum corneum)
147	205858_at	NGFR	NM_002507	nerve growth factor receptor
148	205927_s_at	CTSE	NM_001910	cathepsin E
149	205980_s_at	ARHGAP8	NM_015366	Rho GTPase activating protein 8
150	206002_at	GPR64	NM_005756	G protein-coupled receptor 64
151	206114_at	EPHA4	NM_004438	EphA4
152	206390_x_at		NM_002619	platelet factor 4
153	206594_at	KIAA0135	NM_015148	KIAA0135 protein
154	206595_at	CST6	NM_001323	cystatin EM
155	206714_at	ALOX15B	NM_001141	arachidonate 15-lipoxygenase, second type
156	206757_at	PDE5A	NM_001083	phosphodiesterase 5A, cGMP-specific
157	206866_at	CDH4	NM_001794	cadherin 4, type 1, R-cadherin (retinal)
158	206884_s_at	SCEL	NM_003843	sciellin
159	206912_at	FOXE1	NM_004473	forkhead box E1(thyroid transcription factor2)
160	207111_at		NM_001974	egf-like module cont., mucin-like, hormone rec-like seq 1
161	207144_s_at	CITED1	NM_004143	Cbpp300-interacting transactivator, with GluAsp-rich carboxy-terminal domain, 1
162	207173_s_at		NM_001797	OB-cadherin-1
163	207674_at	FCAR	NM_002000	Fc fragment of IgA
164	207695_s_at	IGSF1	NM_001555	immunoglobulin superfamily, member 1
165	207795_s_at	CD94	AB009597
166	207826_s_at	ID3	NM_002167	inhibitor of DNA binding 3, dominant negative protein
167	207923_s_at	PAX8	NM_013953	paired box gene 8
168	208396_s_at	PDE1A	NM_005019	phosphodiesterase 1A, calmodulin-dependent
169	208451_s_at	C4B	NM_000592	complement component 4B
170	208712_at	PRAD1	M73554	cyclinD1
171	208747_s_at		NM_001734	subcomponent C1s, α- and β-chains
172	209021_x_at		BC001331	Similar to KIAA0652 gene product
173	209035_at		NM_002391	midkine
174	209071_x_at		AF159570	regulator of G-protein signalling 5
175	209079_x_at		AF152318	protocadherin gamma A1
176	209173_at		NM_006408	putative secreted protein XAG
177	209208_at		NM_004870	clone 015e11 My008 protein
178	209228_x_at		NM_006765	Putative prostate cancer tumor suppressor
179	209270_at	LAMB3	NM_000228	laminin S B3 chain
180	209280_at		NM_006039	chromosome 17 unknown product mRNA
181	209291_at		NM_001546	inhibitor of DNA binding 4, dominant negative helix-loop-helix protein
182	209297_at	ITSN	AF114488	intersectin short isoform
183	209335_at		AI281593
184	209365_s_at	ECM1	NM_004425	extracellular matrix protein 1
185	209386_at		AI346835	transmembrane 4 superfamily member 1
186	209485_s_at	ORP1	AF274714	oxysterol-binding protein-related protein
187	209496_at		BC000069	retinoic acid receptor responder 2
188	209505_at		NM_005654	nuclear receptor subfam 2, group F, mem 1
189	209506_s_at		BC004154	nuclear receptor subfam 2, group F, mem 1
190	209529_at		AF047760	phosphatidic acid phosphohydrolase type-2c
191	209596_at		AF245505	adlican
192	209598_at	KIAA0883	AB020690	KIAA0883 protein
193	209652_s_at		BC001422	Similar to placental growth factor, vascular endothelial growth factor-related protein
194	209691_s_at		BC003541	hypothetical protein FLJ10488
195	209739_s_at		AI814551	GS2 gene
196	209772_s_at		X69397	cell surface antigen
197	209781_s_at	T-Star	AF069681	T-Star
198	209792_s_at		BC002710	kallikrein 10
199	209810_at	SP-B	J02761	pulmonary surfactant-associated protein B
200	209897_s_at		AF055585	neurogenic extracellular slit protein Slit2
201	209924_at		AB000221	small inducible cytokine subfamily A (Cys-mem18, pulmonary/activation-reg
202	209946_at		U58111	FLT4 ligand
203	209990_s_at		NM_005458	GABA-B receptor
204	210051_at	CAMP-GEFI	U78168	cAMP-regulated guanine nucleotide exchange factor I
205	210055_at		NM_000369	thyroid stimulating hormone receptor
206	210072_at		NM_006274	beta chemokine Exodus-3
207	210078_s_at		L39833	K+ channel beta subunit
208	210096_at		NM_000779	lung cvtochrome P450 (IV subfamily) BI
209	210298_x_at	FHL1	AF098518	four and 1/2 LIM domains1 protein isoform B
210	210321_at		M36118	cytotoxin serine protease-C
211	210342_s_at	TPO	NM_000547	thyroid peroxidase
212	210372_s_at	TPD52L2	AF208012	tumor protein D52-like 2
213	210397_at		U73945	beta-defensin-1
214	210401_at		U45448	P2x1 receptor
215	210471_s_at		U33428	K+ channel β 1a subunit mRNA, alt spliced
216	210473_s_at	p58GTA	M37712	galactosyltransferase assoc protein kinase
217	210605_s_at		BC003610	Similar to milk fat globule-EGF factor 8
218	210640_s_at	GPCR-Br	U63917	G protein coupled receptor
219	210660_at	LIR-6	AF025529	leucocyte immunoglobulin-like receptor-6b
220	210727_at		NM_001741	Calcitonin, calcitonincalcitonin-rel polypeptide, α
221	210762_s_at	HP	NM_006094	HP protein
222	210809_s_at		D13665	osf-2 mRNA for osteoblast specific factor 2
223	210827_s_at	ESE-1	U73844	epithelial-specific transcription factor ESE-1 a
224	211100_x_at		U82278	immunoglobulin-like transcript 1c
225	211101_x_at		U82276	immunoglobulin-like transcript 1a
226	211102_s_at		U82277	immunoglobufin-like transcript 1b
227	211161_s_at		AF130082	collagen, type III, alpha 1
228	211217_s_at		AF051426	slow delayed rectifier channel subunit
229	211538_s_at		U56725	heat shock 70kD protein 2
230	211564_s_at		BC003096	Similar to LIM domain protein
231	211679_x_at	GABBR2	AF095784	GABA-B receptor R2
232	211813_x_at		AF138303	decorin D
233	211959_at	IGFBP5	AW007532	insulin-like growth factor binding protein 5
234	211964_at		AK025912	type IV collagen alpha (2) chain
235	212067_s_at		AL573058	complement component 1, r subcomponent
236	212253_x_at	KIAA0728	BG253119	KIAA0728 protein
237	212294_at		BG111761	DKFZp586B0918
238	212328_at	KIAA1102	AB029025	KIAA1102 protein
239	212344_at	KIAA1077	AW043713	KIAA1077 protein
240	212353_at	KIAA1077	AI479175	KIAA1077 protein
241	212354_at	KIAA1077	BE500977	KIAA1077 protein
242	212464_s_at	FN	X02761	fibronectin precursor
243	212724_at		NM 005168	ras homolog gene family, member E
244	212738_at		AV717623
245	212775_at		AI978623	KIAA0657 protein
246	212803_at		NM_005967	NGFI-A binding protein 2
247	212806_at	KIAA0367	AL138349	KIAA0367 protein
248	212850_s_at		AA584297	low density lipoprotein receptor-rel protein 4
249	212865_s_at		BF449063	collagen, type XIV, alpha 1 (undulin)
250	212912_at		AI992251	Ribosomal pro S6 kinase, 90 kDa, polypep 2
251	212992_at		AI935123
252	213029_at		AL110126	DKFZp564H1916
253	213306_at		AA917899	multiple PDZ domain protein
254	213381_at		N91149	DKFZp586M2022
255	213423_x_at		AI884858	Putative prostate cancer tumor suppressor
256	213553_x_at		W79394	apolipoprotein C-I
257	213668_s_at		AI989477	SRY (sex determining region Y)-box 4
258	213693_s at		AI610869	mucin 1, transmembrane
259	213800_at		X04697	complement factor H 38-kDa N-term frag
260	213904_at		AL390170	DKFZp547E184
261	213924_at	FLJ11585	BF476502	hypothetical protein FLJ11585
262	214023_x_at		AL533838	tubulin, beta polypeptide
263	214175_x_at		AI254547	LIM domain protein
264	214239_x_at	Mel-18	AI560455	Zinc finger protein 144
265	214307_at		AI478172	homogentisate 1,2-dioxygenase
266	214434_at	KIAA0417	AB007877	KIAA0417 gene product
267	214632_at		NM_003872	neuropilin 2
268	214680_at		BF674712	neurotrophic tyrosine kinase receptor 2
269	214702_at	MSF-FN70	AJ276395	migration stimulation factor FN70
270	214763_at	FLJ13875	AK023937	FLJ13875
271	214803_at		BF344237	DKFZp564N1116
272	214955_at		AI912086	DNA seq clone 1170K4 chrom 22q12.2-13.1
273	214977_at	FLJ13790	AK023852	FLJ13790
274	215016_x_at	KIAA0728	BC004912	KIAA0728 protein
275	215034_s_at	FLJ13302	AI189753	FLJ13302
276	215076_s_at	FLJ11428	AU144167	FLJ11428
277	215243_s_at	GJB3	AF099730	connexin 31
278	215388_s_at		X56210	complement Factor H-related protein 1
279	215442_s_at		BE740743	thyroid stimulating hormone receptor
280	215443_at		BE740743	thyroid stimulating hormone receptor
281	215506_s_at		AK021882	highly sim to putative tumor sup NOEY2
282	215536_at	LMP7,	X87344	DMA, DMB, HLA-Z1, IPP2, LMP2, TAP1, TAP2, DOB, DQB2 and RING8, 9, 13, 14 genes
283	216356_x_at	KIAA0734	AB018277	KIAA0734 protein
284	216470_x_at		AF009664	T cell receptor β locus, 3 trypsinogen repeats
285	216569_at	FABP3-ps	U72237	fatty acid-binding protein pseudogene
286	217546_at		R06655
287	217561_at		BF447272
288	217592_at		AV684859
289	217767_at		NM 000064
290	217820_s_at	FLJ10773	NM_018212	hypothetical protein FLJ10773
291	217875_s_at	TMEPAI	NM_020182	transmem, prostate androgen induced RNA
292	218002_s_at	SCYB14	NM_004887	small inducible cytokine subfam B (Cys-X-mem 14 (BRAK)
293	218182_s_at	CLDN1	NM_021101	claudin 1
294	218353_at		NM_025226	MSTP032 protein
295	218368_s_at	FN14	NM_016639	type I transmembrane protein Fn14
296	218418_s_at		NM_015493	DKFZP434N161
297	218469_at	CKTSF1B1	NM_013372	Cvs knot superfamily 1, BMP antagonist 1
298	218537_at	FLJ20568	NM_017885	hypothetical protein FLJ20568
299	218546_at	FLJ14146	NM_024709	hypothetical protein FLJ14146
300	218613_at		NM_018422	hypothetical protein DKFZp761K1423
301	218653_at	SLC25A15	NM_014252	solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 15
302	218691_s at	RIL	NM_003687	reversion-induced LIM protein
303	218844_at	FLJ20920	NM_025149	hypothetical protein FLJ20920
304	218856_at	LOC51323	NM_016629	hypothetical protein LOC51323
305	218952_at	SAAS	NM_013271	granin-like neuroendocrine peptide precursor
306	218960_at	TMPRSS4	NM_016425	transmembrane protease, serine 4
307	219010_at	FLJ10901	NM_018265	hypothetical protein FLJ10901
308	219127_at	MGC11242	NM_024320	hypothetical protein MGC11242
309	219191_s_at	BIN2	NM_016293	bridging integrator 2
310	219195_at	PPARGC1	NM_013261	peroxisome proliferative activated receptor, y, coactivator 1
311	219211_at	USP18	NM_017414	ubiquitin specific protease 18
312	219277_s_at	FLJ10851	NM_018245	hypothetical protein FLJ10851
313	219331_s_at	FLJ10748	NM_018203	hypothetical protein FLJ10748
314	219416_at	CSR1	NM_016240	CSR1 protein
315	219436_s_at	LOC51705	NM_016242	endomucin-2
316	219440_at	RAI2	NM_021785	retinoic acid induced 2
317	219463_at	HS1119D91	NM_012261	sim to S68401 (cattle) glucose induced gene
318	219476_at	MGC4309	NM_024115	hypothetical protein MGC4309
319	219525_at	FLJ10847	NM_018242	hypothetical protein FLJ10847
320	219527_at	FLJ20605	NM_017898	hypothetical protein FLJ20605
321	219561_at	LOC51226	NM_016429	COPZ2 for nonclathrin coat protein zeta-COP
322	219596_at	LOC56906	NM_020147	hyp protein from EUROIMAGE 511235
323	219597_s_at	DUOX1	NM_017434	dual oxidase 1
324	219743_at	HEY2	NM_012259	hairyenhancer-of-split rel with YRPW motif 2
325	219749_at	FLJ20967	NM_022071	hypothetical protein FLJ20967
326	219836_at	MGC10796	NM_024508	hypothetical protein MGC10796
327	219855_at	FLJ10628	NM_018159	hypothetical protein FLJ10628
328	219856_at	MGC2742	NM_023938	hypothetical protein MGC2742
329	219926_at	POP3	NM_022361	popeye protein 3
330	219932_at	VLCS-H 1	NM_014031	v long-chain acyl-CoA synthetase homolog 1
331	219958_at	FLJ11190	NM_018354	hypothetical protein FLJ11190
332	220034_at		NM_007199	interleukin-1 receptor-associated kinase M
333	220108_at	GNA14	NM_004297	guanine nucl binding protein (G protein), α 14
334	220332_at	CLDN16	NM_006580	claudin 16
335	220595_at	DKFZp434B0 417	NM_013377	hypothetical protein DKFZp434B0417
336	220751_s_at	C5ORF4	NM_016348	chromosome 5 open reading frame 4
337	221009_s_at	PGAR	NM_016109	PPAR(gamma) angiopoietin related protein
338	221073_s_at	NOD1	NM_006092	caspase recruitment domain 4
339	221147_x_at	WWOX	NM_018560	WW domain-containing oxidoreductase
340	221266_s_at	LOC81501	NM_030788	DC-specific transmembrane protein
341	221270_s_at	TGT	NM_031209	tRNA-guanine transglycosylase
342	221489_s_at		AF227517	sprouty (Drosophila) homolog 4
343	221577_x_at		AF003934	prostate differentiation factor
344	221636_s_at		AL136931	DKFZp586G2122
345	221701_s_at		AF352728	STRA6 isoform 1 mRNA, alternatively spliced
346	221724_s_at		AF200738	C-type lectin DDB27 short form
347	221795_at		AI346341	Similar to hypothetical protein FLJ20093
348	221796_at		AA707199	Similar to hypothetical protein FLJ20093
349	221799_at	KIAA1402	AB037823	KIAA1402 protein
350	221870_at		AI417917	FLJ22356 fis
351	221900_at		AI806793	collagen, type VIII, alpha 2
352	221928_at		AI057637	Weakly sim to 2109260A B cell growth factor
353	221959_at		BE672313	highly sim to HSU79298 Human clone 23803
354	32128_at		Y13710	alternative activated macrophage specific CC chemokine 1
355	37004_at	SP-B	J02761	pulmonary surfactant-associated protein B
356	37117_at		Z83838	GTPase-activating protein sim to rhoGAP protein. ribosomal protein L6 pseudogene
357	37152_at		L07592	peroxisome proliferator activated receptor
358	37408_at	KIAA0709	AB014609	KIAA0709 protein
359	38691_s at	SP5	J03553	pulmonary surfactant protein
360	45297_at		AI417917
361	63305_at		D81792
362	823_at		HSU84487	CX3C chemokine precursor, alt spliced
363	91920_at		AI205180
364				SGENE forward primer
365				SGENE reverse primer
366				SGENE probe
367				TESTICAN1 forward primer
368				TESTICAN1 reverse primer
369				TESTICAN1 probe
370				GABRE forward primer
371				GABRE reverse primer
372				GABRE probe
373				CDH3 forward primer
374				CDH3 reverse primer
375				CDH3 probe
376				FN1 forward primer
377				FN1 reverse primer
378				FN1 probe
379				TPO-1 forward primer
380				TPO-1 reverse primer
381				TPO-1 probe
382				TPO-2 forward primer
383				TPO-2 reverse primer
384				TPO-2 probe
385				KCNAB1-1 forward primer
386				KCNAB1-1 reverse primer
387				KCNAB1-1 probe
388				KCNAB1-2 forward primer
389				KCNAB1-2 reverse primer
390				KCNAB1-2 probe
391				FABP4-1 forward primer
392				FABP4-1 reverse primer
393				FABP4-1 probe
394				FABP4-2 forward primer
395				FABP4-2 reverse primer
396				FABP4-2 probe
397				DOI1-1 forward primer
398				DOI1-1 reverse primer
399				DOI1-1 probe
400				DOI1-2 forward primer
401				DOI1-2 reverse primer
402				DOI1-2 probe
403				B-ACTIN forward primer
404				B-ACTIN reverse primer
405				B-ACTIN probe
406				GOLGIN67 forward primer
407				GOLGIN67 reverse primer
408				GOLGIN67 probe
409				PAX8 forward primer
410				PAX8 reverse primer
411				PAX8 probe
412				HERC forward primer
413				HERC reverse primer
414				HERC probe
415				DDIT3 forward primer
416				DDIT3 reverse primer
417				DDIT3 probe
418				ITM1 forward primer
419				ITM1 reverse primer
420				ITM1 probe
421				C1OF24 forward primer
422				C1OF24 reverse primer
423				C1OF24 probe
424				MOT8 forward primer
425				MOT8 reverse primer
426				MOT8 probe
427				ARG2 forward primer
428				ARG2 reverse primer
429				ARG2 probe

Claims

1. A method of diagnosing thyroid cancer, testing indeterminate thyroid fine needle aspirate (FNA) thyroid nodule samples, or of differentiating between thyroid carcinoma and benign thyroid diseases in a biological sample obtained from a patient; comprising the step of measuring the expression levels in the sample of genes encoding mRNA corresponding to SEQ ID NOs: 199, 207, 255 and 354; wherein the gene expression levels above or below pre-determined cut-off levels are indicative of thyroid cancer or of thyroid carcinoma.

2. The method of claim 1 wherein the sample was prepared by a method selected from the group consisting of fine needle aspiration, bulk tissue preparation (for example, from a biopsy or a surgical specimen) and laser capture microdissection.

3. The method of claim 1 further comprising measuring

a) the expression level of at least one gene encoding mRNA:

i. corresponding to SEQ ID NOs: 142, 219 and 309; and/or

ii. corresponding to SEQ ID NOs: 9, 12 and 18; or

b) the expression level of at least one gene constitutively expressed in the sample.

4. The method of claim 1 wherein the comparison of expression patterns is conducted with pattern recognition methods, which can optionally include the use of a Cox proportional hazards analysis.

5. The method of claim 1 wherein the pre-determined cut-off levels

a) are at least about 1.5-fold over- or under- expression in the sample relative to benign cells or normal tissue; or

b) have at least a statistically significant p-value over-expression in the sample having thyroid carcinoma cells relative to benign cells or normal tissue, wherein optionally the p-value is less than about 0.05.

6. The method of claim 1 wherein gene expression

a) is measured on a microarray or gene chip

b) is determined by nucleic acid amplification conducted by polymerase chain reaction (PCR) of RNA extracted from the sample; or

c) is detected by measuring or detecting a protein encoded by the gene; or

d) is detected by measuring a characteristic of the gene, which may optionally be selected from the group consisting of DNA amplification, methylation, mutation and allelic variation.

7. The method of claim 6(a) wherein the microarray is a cDNA array or an oligonucleotide array which may optionally comprise one or more internal control reagents.

8. The method of claim 6(b) wherein said PCR is reverse transcription polymerase chain reaction (RT-PCR).

9. The method of claim 8, wherein the RT-PCR further comprises one or more internal controls, for example a method of detecting PAX8 gene expression which may optionally be measured using SEQ ID NOs: 409-411.

10. The method of claim 6(c) wherein the protein is detected by an antibody specific to the protein.

11. A composition comprising at least one probe set consisting of SEQ ID NOs: 199, 207, 255 and 354.

12. A kit for conducting an assay to determine thyroid carcinoma prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes encoding mRNA corresponding to SEQ ID NOs: 199, 207, 255 and 354, wherein the kit optionally further comprises reagents for conducting a microarray analysis or a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

13. The use of a microarray or gene chip for performing the method of claim 1.

14. The use of claim 13 wherein the microarray or gene chip comprises isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes encoding mRNA corresponding to SEQ ID NOs: 199, 207, 255 and 354;
where the combination is sufficient to characterize thyroid carcinoma or risk of relapse in a biological sample.

15. The use of claim 14 wherein:

a) the measurement or characterization is at least about 1.5-fold over- or under-expression; or

b) the microarray or gene chip comprises a cDNA array or an oligonucleotide array; or

c) the microarray or gene chip comprises one or more internal control reagents.

Drawing